Microorganisms for therapy

ABSTRACT

Therapeutic methods and vaccinia virus therefor are provided. The viruses are designed so that they accumulate in immunoprivileged tissues and cells, such as in tumors and other proliferating tissue and in inflamed tissues, compared to other tissues, cells and organs, so that they exhibit relatively low toxicity to host organisms. Combinations of the viruses and anti-cancer agents and uses thereof for treating cancer also are provided.

RELATED APPLICATIONS

This application is a continuation of allowed U.S. application Ser. No.12/589,694, to Aladar A. Szalay; Tatyana Timiryasova; Yong A. Yu; QianZhang, filed Oct. 26, 2009, entitled “MICROORGANISMS FOR THERAPY,” whichis a continuation of U.S. application Ser. No. 11/796,028, to Aladar A.Szalay; Tatyana Timiryasova; Yong A. Yu; Qian Zhang, filed on Apr. 25,2007, entitled “MICROORGANISMS FOR THERAPY,” which is a divisional ofU.S. application Ser. No. 10/872,156, to Aladar A. Szalay, TatyanaTimiryasova, Yong A. Yu and Qian Zhang, filed on Jun. 18, 2004, entitled“MICROORGANISMS FOR THERAPY,” which claims the benefit of priority under35 U.S.C. §119(a) to each of EP 03 013 826.7, filed 18 Jun. 2003,entitled “RECOMBINANT VACCINIA VIRUSES USEFUL AS TUMOR-SPECIFIC DELIVERYVEHICLE FOR CANCER GENE THERAPY AND VACCINATION;” EP 03 018 478.2, filed14 Aug. 2003, entitled “METHOD FOR THE PRODUCTION OF A POLYPEPTIDE, RNAOR OTHER COMPOUND IN TUMOR TISSUE;” and EP 03 024 283.8, filed 22 Oct.2003, entitled “USE OF A MICROORGANISM OR CELL TO INDUCEAUTOIMMUNIZATION OF AN ORGANISM AGAINST A TUMOR.” The subject matter ofeach of these applications is incorporated by reference in its entirety.

This application is also a continuation of U.S. application Ser. No.11/796,028, to Aladar A. Szalay; Tatyana Timiryasova; Yong A. Yu; QianZhang, filed on Apr. 25, 2007, entitled “MICROORGANISMS FOR THERAPY,”which is a divisional of U.S. application Ser. No. 10/872,156, to AladarA. Szalay, Tatyana Timiryasova, Yong A. Yu and Qian Zhang, filed on Jun.18, 2004, entitled “MICROORGANISMS FOR THERAPY,” which claims thebenefit of priority under 35 U.S.C. §119(a) to each of EP 03 013 826.7,filed 18 Jun. 2003, entitled “RECOMBINANT VACCINIA VIRUSES USEFUL ASTUMOR-SPECIFIC DELIVERY VEHICLE FOR CANCER GENE THERAPY ANDVACCINATION;” EP 03 018 478.2, filed 14 Aug. 2003, entitled “METHOD FORTHE PRODUCTION OF A POLYPEPTIDE, RNA OR OTHER COMPOUND IN TUMOR TISSUE;”and EP 03 024 283.8, filed 22 Oct. 2003, entitled “USE OF AMICROORGANISM OR CELL TO INDUCE AUTOIMMUNIZATION OF AN ORGANISM AGAINSTA TUMOR.” The subject matter of each of these applications isincorporated by reference in its entirety.

This application also is related to International Application Serial No.PCT/US04/19866, filed on Jun. 18, 2004. This application also is relatedto U.S. application Ser. No. 10/866,606, filed Jun. 10, 2004, entitled“LIGHT EMITTING MICROORGANISMS AND CELLS FOR DIAGNOSIS AND THERAPY OFTUMORS,” which is a continuation of U.S. application Ser. No.10/189,918, filed Jul. 3, 2002, entitled “LIGHT EMITTING MICROORGANISMSAND CELLS FOR DIAGNOSIS AND THERAPY OF TUMORS”; U.S. application Ser.No. 10/849,664, filed May 19, 2004, entitled, “LIGHT EMITTINGMICROORGANISMS AND CELLS FOR DIAGNOSIS AND THERAPY OF DISEASESASSOCIATED WITH WOUNDED OR INFLAMED TISSUE” which is a continuation ofU.S. application Ser. No. 10/163,763, filed Jun. 5, 2002, entitled“LIGHT EMITTING MICROORGANISMS AND CELLS FOR DIAGNOSIS AND THERAPY OFDISEASES ASSOCIATED WITH WOUNDED OR INFLAMED TISSUE”; International PCTApplication WO 03/014380, filed Jul. 31, 2002, entitled “MICROOROGANISMSAND CELLS FOR DIAGNOSIS AND THERAPY OF TUMORS;” PCT Application WO03/104485, filed Jun. 5, 2003, entitled, “LIGHT EMITTING MICROORGANISMSAND CELLS FOR DIAGNOSIS AND THERAPY OF DISEASES ASSOCIATED WITH WOUNDEDOR INFLAMED TISSUE;” EP Application No. 01 118 417.3, filed Jul. 31,2001, entitled “LIGHT EMITTING MICROORGANISMS AND CELLS FOR TUMOURDIAGNOSIS/THERAPY;” EP Application No. 01 125 911.6, filed Oct. 30,2001, entitled “LIGHT EMITTING MICROORGANISMS AND CELLS FOR DIAGNOSISAND THERAPY OF TUMORS;” EP Application No. 02 794 632.6, filed Jan. 28,2004, entitled “VACCINIA VIRUS FOR DIAGNOSIS AND THERAPY OF TUMORS;” andEP Application No. 02 012 552.2, filed Jun. 5, 2002, entitled “LIGHTEMITTING MICROORGANISMS AND CELLS FOR DIAGNOSIS AND THERAPY OF DISEASESASSOCIATED WITH WOUNDED OR INFLAMED TISSUE.” The subject matter of eachof these applications is incorporated by reference in its entirety.

FIELD OF THE INVENTION

Vaccines that contain attenuated or modified microorganisms, includingmicrobes and cells, and methods for preparing the microorganisms andvaccines are provided. In particular, modified bacteria, eukaryoticcells and viruses are provided and methods of use thereof for treatmentof proliferative and inflammatory disorders and for production ofproducts in tumors are provided.

BACKGROUND

In the late 19th century, a variety of attempts were made to treatcancer patients with microorganisms. One surgeon, William Coley,administered live Streptococcus pyogenes to patients with tumors withlimited success. In the early 20th century, scientists documentedvaccinia viral oncolysis in mice, which led to administration of severallive viruses to patients with tumors from the 1940s through the 1960s.These forays into this avenue of cancer treatment were not successful.

Since that time, a variety of genetically engineered viruses have beentested for treatment of cancers. In one study, for example, nude micebearing nonmetastatic colon adenocarcinoma cells were systemicallyinjected with a WR strain of vaccinia virus modified by having avaccinia growth factor deletion and an enhanced green fluorescenceprotein inserted into the thymidine kinase locus. The virus was observedto have antitumor effect, including one complete response, despite alack of exogenous therapeutic genes in the modified virus (McCart et al.(2001) Cancer Res 1:8751-8757). In another study, vaccinia melanomaoncolysate (VMO) was injected into sites near melanoma positive lymphnodes in a Phase III clinical trial of melanoma patients. As a control,New York City Board of Health strain vaccinia virus (VV) wasadministered to melanoma patients. The melanoma patients treated withVMO had a survival rate better than that for untreated patients, butsimilar to patients treated with the VV control (Kim et al. (2001)Surgical Oncol 10:53-59).

Other studies have demonstrated limited success with this approach. Thistherapy is not completely effective, particularly for systemicallydelivered viruses or bacteria. Limitations on the control of microbialvehicle function in vivo result in ineffective therapeutic results aswell as raising safety concerns. It would be desirable to improve thistype of therapy or to develop more effective approaches for treatmentsof neoplastic disease. Therefore, among the objects herein, it is anobject to provide therapeutic methods and microorganisms for thetreatment of neoplastic and other diseases.

SUMMARY

Provided herein are therapeutic methods and microorganisms, includingviruses, bacteria and eukaryotic cells, for uses in the methods for thetreatment of neoplastic diseases and other diseases. Diseases fortreatment are those in which the targeted tissues and/or cells areimmunoprivileged in that they, and often the local environment thereof,somehow escape or are inaccessible to the immune system. Such tissuesinclude tumors and other tissues and cells involved in otherproliferative disorders, wounds and other tissues involved ininflammatory responses. The microorganisms, which include bacterialcells, viruses and mammalian cells, are selected or are designed to benon-pathogenic and to preferentially accumulate in the immunoprivilegedtissues. The microorganisms, once in the tissues or cells or vicinitythereof, affect the cell membranes of the cells in such tissues so thatthey become leaky or lyse, but sufficiently slowly so that the targetedcells and tumors leak enough antigen or other proteins for a timesufficient to elicit an immune response.

The microorganisms are administered by any route, including systemicadministration, such as i.v. or using oral or nasal or other deliverysystems that direct agents to the lymphatics. In exemplary methods, themicroorganisms are used to treat tumors and to prevent recurrence andmetastatic spread. Exemplary microorganisms include highly attenuatedviruses and bacteria, as well as mammalian cells. The microorganisms areoptionally modified to deliver other products, including othertherapeutic products to the targeted tissues.

When the microorganisms are administered to a host that contains tumors,the tumors in the host essentially become antigen and protein factories.This can be exploited so that the tumors can be used to produce proteinsor other cellular products encoded by or produced by the microorganisms.In addition, the host sera can be harvested to isolate antibodies toproducts produced by the microorganisms as well as the tumor cells.Hence also provided are methods for producing gene products byadministering the microorganisms to an animal, generally a non-humananimal, and harvesting the tumors to isolate the product. Also providedare methods for producing antibodies to selected proteins or cellproducts, such as metabolites or intermediates, by administering amicroorganism that expresses or produces the protein or other product toa host, typically a non-human host; and harvesting serum from the hostand isolating antibodies that specifically bind to the protein or otherproduct.

Thus provided are methods and microorganisms for elimination ofimmunoprivileged cells or tissues, particularly tumors. The methodsinclude administration, typically systemic administration, with amicroorganism that preferentially accumulates in immunoprivileged cells,such as tumor cells, resulting in leakage proteins and other compounds,such as tumor antigens, resulting in vaccination of the host againstnon-host proteins and, such as the tumor antigens, providing forelimination of the immunoprivileged cells, such as tumor cells, by thehost's immune system. The microorganisms are selected not for theirability to rapidly lyse cells, but rather for the ability to accumulatein immunoprivileged cells, such as tumors, resulting in a leakage ofantigens in a sufficient amount and for a sufficient time to elicit animmune response.

Hence provided are uses of microorganisms or cells containingheterologous DNA, polypeptides or RNA to induce autoimmunization of anorganism against a tumor. In particular, the microorganisms are selectedor designed to accumulate in tumors and to accumulate very little, if atall (to be non-toxic to the host) in non-tumorous cells, tissues ororgans, and to in some manner result in the tumor cell lyses or cellmembrane disruption such that tumor antigens leak. Exemplary of suchmicroorganisms are the LIVP-derived vaccinia virus and the bacteriadescribed herein and also mammalian cells modified to target the tumorsand to disrupt the cells membrane. The microorganisms can be modified toexpress heterologous products that mediate or increase the leakage ofthe tumor cell antigens and/or that are therapeutic, such as anti-tumorcompounds.

Also provided are methods for production of antibodies against a tumorby (a) injecting a microorganism or cell containing a DNA sequenceencoding a desired polypeptide or RNA into an organism bearing a tumorand (b) isolating antibodies against the tumor.

Provided are attenuated microorganisms that accumulate inimmunoprivileged tissues and cells, such as tumor cells, but do notaccumulate to toxic levels in non-targeted organs and tissues, and thatupon administration to an animal bearing the immunoprivileged tissuesand cells, result in autoimmunity, such as by production of anti-tumor(or anti-tumor antigen) antibodies against the immunoprivileged cells orproducts thereof. The microorganisms are selected or produced to renderthe immunoprivileged cells leaky, such as by a slow lysis or apoptoticprocess. The goal is to achieve such leakiness, but to not lyse thecells so rapidly that the host cannot mount an immune response.

Uses of and methods of use of the microorganisms for eliminatingimmunoprivileged tissues and cells are provided. The microorganismsoptionally include reporter genes and/or other heterologous nucleicacids that disrupt genes in the microorganism and can also encode andprovide therapeutic products or products, such as RNA, including RNAi,that alter gene and/or protein expression in the cells or tissues wherethe microorganism accumulates. Among the viruses provided are attenuatedpox viruses that contain a modified TK and HA gene and a modified F3gene or locus that corresponds to the F3 gene in vaccinia. Inparticular, provided are recombinant vaccinia viruses that contain amodified TK and HA gene and optionally a modified F3 gene or locus,wherein the resulting virus does not accumulate to toxic levels innon-targeted organs. Vaccinia viruses where the TK gene and F3 gene aremodified and vaccinia viruses where the HA and F3 gene are modified, andviruses where all three genes are modified are provided. Modificationincludes inactivation by insertion, deletion or replacement of one ormore nucleotide bases whereby an activity or product of the virus isaltered. Included among the alterations is insertion of heterologousnucleic acid, such as therapeutic protein-encoding nucleic acids.

In exemplary embodiments, the vaccinia viruses are Lister strainviruses, particularly LIVP strain viruses (LIVP refers to the Listervirus from the Institute of Viral Preparations, Moscow, Russia, theoriginal source for this now widely disseminated virus strain).Modifications include modification of the virus at the unique NotI sitein the locus designed F3. In particular, the modification can be atposition 35 of the F3 locus (gene) or at position 1475 inside of theHindIII-F fragment of vaccinia virus DNA strain LIVP.

The heterologous nucleic acid can include regulatory sequencesoperatively linked to the nucleic acid encoding the protein. Regulatorysequences include promoters, such as the vaccinia virus early/latepromoter p7.5 and an early/late vaccinia pE/L promoter. The heterologousnucleic acid in the microorganism can encode a detectable protein or aproduct capable of inducing a detectable signal. Inclusion of detectableprotein or a product that can generate a detectable signal permitsmonitoring of the distribution of the administered microorganism as wellas monitoring therapeutic efficacy, since the microorganism will beeliminated when the immunoprivileged cells are eliminated.

Host cells containing the recombinant viruses, such as the triple mutantvaccinia virus exemplified herein are provided. Also contemplated aretumor cells that contain any of the microorganisms provided herein orused in the methods.

Pharmaceutical compositions containing the microorganisms in apharmaceutically acceptable vehicle for use in the methods herein areprovided. The pharmaceutical compositions can be formulated for any modeof administration, including, but not limited to systemicadministration, such as for intravenous administration or is formulated.The compositions can contain a delivery vehicle, such as a lipid-basedcarrier, including liposomes and micelles associated with themicroorganism.

Also provided are methods (and uses of the microorganisms) foreliminating immunoprivileged cells, such as tumor cells in an animal, byadministering the pharmaceutical compositions to an animal, whereby thevirus accumulates in the immunoprivileged cells, thereby mediatingautoimmunization resulting in elimination of the cells or a reduction intheir number.

Therapeutic methods for eliminating immunoprivileged cells or tissues,in an animal, by administering a microorganism to an animal, where themicroorganism accumulates in the immunoprivileged cells; themicroorganism does not accumulate in unaffected organs and tissues andhas low toxicity in the animal; and the microorganism results in leakageof the cell membranes in the immunoprivileged cells, whereby the animalproduces autoantibodies against the cells or products of the cells areprovided. These methods include tumor treatment, treatment forinflammatory conditions, including wounds, and proliferative disorders,including psoriasis, cancers, diabetic retinopathies, restenosis andother such disorders. It is desirable for the microorganisms to notaccumulate in unaffected organs, particularly the ovaries or testes.

The microorganisms attenuated include attenuated viruses, such as poxviruses and other cytoplasmic viruses, bacteria such as vibrio, E. coli,salmonella, streptococcus and listeria and mammalian cells, such asimmune cells, including B cells and lymphocytes, such as T cells, andstem cells.

Also provided are methods for production of a polypeptide or RNA orcompound, such as a cellular product, and uses of the microorganismtherefore are provided. Such methods can include the steps of: (a)administering a microorganism containing nucleic acid encoding thepolypeptide or RNA or producing the product compound to tumor-bearinganimal, where the microorganism accumulates in the immunoprivilegedcells; and the microorganism does not accumulate to toxic levels inorgans and tissues that do not comprise immunoprivileged cells ortissues; (b) harvesting the tumor tissue from the animal; and (c)isolating the polypeptide or RNA or compound from the tumor.

As noted, the microorganisms include eukaryotic cells, prokaryotic cellsand viruses, such as a cytoplasmic virus or an attenuated bacterium or astem cell or an immune cell. The bacterium can be selected from amongattenuated vibrio, E. coli, listeria, salmonella and streptococcusstrains. The microorganism can express or produce detectable products,such as a fluorescent protein (i.e., green, red and blue fluorescentproteins and modified variants thereof), and/or luciferase which, whencontacted with a luciferin produces light, and also can encodeadditional products, such as therapeutic products. In the methods anduses provided herein, the animals can be non-human animals or caninclude humans.

Also provided are methods for simultaneously producing a polypeptide,RNA molecule or cellular compound and an antibody that specificallyreacts with the polypeptide, RNA molecule or compound, by: a)administering a microorganism to a tumor-bearing animal, wherein themicroorganism expresses or produces the compound, polypeptide or RNAmolecule; and b) isolating the antibody from serum in the animal. Themethod optionally includes, after step a) harvesting the tumor tissuefrom the animal; and isolating the polypeptide, RNA molecule or cellularcompound from the tumor tissue.

Also provided are methods for eliminating immunoprivileged cells ortissues in an animal, such as tumor cells, and uses of themicroorganisms therefore by administering at least two microorganisms,wherein the microorganisms are administered simultaneously, sequentiallyor intermittently, wherein the microorganisms accumulate in theimmunoprivileged cells, whereby the animal is autoimmunized against theimmunoprivileged cells or tissues.

Uses of at least two microorganisms for formulation of a medicament forelimination of immunoprivileged cells or tissues, wherein theyaccumulate in the immunoprivileged cells, whereby the animal isautoimmunized against the immunoprivileged cells or tissues areprovided. Combinations containing at least two microorganisms formulatedfor administration to an animal for elimination of immunoprivilegedcells or tissues are provided. Kits containing packaged combinationoptionally with instructions for administration and other reagents areprovided.

Uses of a microorganism encoding heterologous nucleic acid for inducingautoimmunization against products produced in immunoprivileged cells,wherein, when administered, the microorganism accumulates inimmunoprivileged tissues and does not accumulate or accumulates at asufficiently low level in other tissues or organs to be non-toxic to ananimal containing the immunoprivileged tissues are provided.

Methods for the production of antibodies against products produced inimmunoprivileged tissues or cells by: (a) administering a microorganismcontaining nucleic acid encoding a selected protein or RNA into ananimal containing the immunoprivileged tissues or cells; and (b)isolating antibodies against the protein or RNA from the blood or serumof the animal are provided.

Also provided are methods for inhibiting growth of immunoprivilegedcells or tissue in a subject by: (a) administering to a subject amodified microorganism, wherein the modified microorganism encodes adetectable gene product; (b) monitoring the presence of the detectablegene product in the subject until the detectable gene product issubstantially present only in immunoprivileged tissue or cells of asubject; and (c) administering to a subject a therapeutic compound thatworks in conjunction with the microorganism to inhibit growth ofimmunoprivileged cells or tissue or by: (a) administering to a subject amodified microorganism that encodes a detectable gene product; (b)administering to a subject a therapeutic substance that reduces thepathogenicity of the microorganism; (c) monitoring the presence of thedetectable gene product in the subject until the detectable gene productis substantially present only in immunoprivileged tissue or cells of asubject; and (d) terminating or suspending administration of thetherapeutic compound, whereby the microorganism increases inpathogenicity and the growth of the immunoprivileged cells or tissue isinhibited.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic of the various vaccinia strains described in theExamples. Results achieved with the viruses are described in theExamples.

FIG. 2 sets forth a flow chart for a method for producing products, suchas nucleic acid molecules, proteins and metabolic compounds or othercellular products in tumors.

DETAILED DESCRIPTION

A. Definitions

B. Microorganisms for Tumor-Specific Therapy

-   -   1. Characteristics        -   a. Attenuated            -   i. Reduced toxicity            -   ii. Accumulate in immunoprivileged cells and tissues,                such as tumor, not substantially in other organs            -   iii. Ability to Elicit or Enhance Immune Response to                Tumor Cell            -   iv. Balance of Pathogenicity and Release of Tumor                Antigens        -   b. Immunogenicity        -   c. Replication Competent        -   d. Genetic Variants            -   i. Modified Characteristics            -   ii. Exogenous Gene Expression            -   iii. Detectable gene product            -   iv. Therapeutic gene product            -   v. Expressing a superantigen            -   vi. Expressing a gene product to be harvested    -   2. Viruses        -   a. Cytoplasmic viruses            -   i. Poxviruses                -   a. Vaccinia Virus                -   b. Modified Vaccinia Viruses                -   c. The F3 Gene                -   d. Multiple Modifications                -   e. The Lister Strain            -   ii. Other cytoplasmic viruses        -   b. Adenovirus, Herpes, Retroviruses    -   3. Bacteria        -   a. Aerobic bacteria        -   b. Anaerobic bacteria    -   4. Eukaryotic cells

C. Methods for Making an Attenuated Microorganism

-   -   1. Genetic Modifications    -   2. Screening for above characteristics

D. Therapeutic Methods

-   -   1. Administration        -   a. Steps prior to administering the microorganism        -   b. Mode of administration        -   c. Dosage        -   d. Number of administrations        -   e. Co-administrations            -   i. Administering a plurality of microorganisms            -   ii. Therapeutic compounds        -   f. State of subject    -   2. Monitoring        -   a. Monitoring microorganismal gene expression        -   b. Monitoring tumor size        -   c. Monitoring antibody titer        -   d. Monitoring general health diagnostics        -   e. Monitoring coordinated with treatment

E. Methods of Producing Gene Products and Antibodies

-   -   1. Production of Recombinant Proteins and RNA molecules    -   2. Production of Antibodies

F. Pharmaceutical Compositions, combinations and kits

-   -   1. Pharmaceutical Compositions    -   2. Host Cells    -   3. Combinations    -   4. Kits

G. Examples

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, websites and other publishedmaterials referred to throughout the entire disclosure herein, unlessnoted otherwise, are incorporated by reference in their entirety. In theevent that there are a plurality of definitions for terms herein, thosein this section prevail. Where reference is made to a URL or other suchidentifier or address, it is understood that such identifiers can changeand particular information on the interne can come and go, butequivalent information is known and can be readily accessed, such as bysearching the internet and/or appropriate databases. Reference theretoevidences the availability and public dissemination of such information.

As used herein, microorganisms refers to isolated cells or viruses,including eukaryotic cells, such as mammalian cells, viruses andbacteria. The microorganisms are modified or selected for their abilityto accumulate in tumors and other immunoprivileged cells and tissues,and to minimize accumulation in other tissues or organs. Accumulationoccurs by virtue of selection or modification of the microorganisms forparticular traits or by proper selection of cells. The microorganism canbe further modified to alter a trait thereof and/or to deliver a geneproduct. The microorganisms provided herein are typically modifiedrelative to wild type to exhibit one or more characteristics such asreduced pathogenicity, reduced toxicity, preferential accumulation intumors relative to normal organs or tissues, increased immunogenicity,increased ability to elicit or enhance an immune response to tumorcells, increased lytic or tumor cell killing capacity, decreased lyticor tumor cell killing capacity.

As used herein, immunoprivileged cells and tissues refer to cells andtissues, such as solid tumors and wounded tissues, which are sequesteredfrom the immune system. Generally administration of a microorganismelicits an immune response that clears the microorganism;immunoprivileged sites, however, are shielded or sequestered from theimmune response, permitting the microorganisms to survive and generallyto replicate. Immunoprivileged tissues include inflamed tissues, such aswounded tissues, and proliferating tissues, such as tumor tissues.

As used herein, “modified” with reference to a gene refers to a deletedgene, or a gene encoding a gene product having one or more truncations,mutations, insertions or deletions, typically accompanied by at least achange, generally a partial loss of function.

As used herein F3 gene refers to a gene or locus in a virus, such as avaccinia virus, that corresponds to the F3 gene of vaccinia virus strainLIVP. This includes the F3 gene of any vaccinia virus strain or poxvirusencoding a gene product having substantially the same or at least arelated biological function or locus in the genome. F3 genes encompassedherein typically have at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 85%,at least about 90%, at least about 93%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identity along the full length of the sequence of nucleotides set forthin SEQ ID NO:1. The proteins encoded by F3 genes encompassed hereintypically have at least about 50%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 85%, atleast about 90%, at least about 93%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%identity to the sequence of amino acids set forth SEQ ID NO:2 along thefull-length sequence thereof. Also included are corresponding loci inother viruses that when modified or eliminated result in reducedtoxicity and/or enhanced accumulation in tumors (compared tonon-tumorous cells, tissues and organs). The corresponding loci in otherviruses equivalent to the F3 gene in LIVP can be determined by thestructural location of the gene in the viral genome: the LIVP F3 gene islocated on the HindIII-F fragment of vaccinia virus between open readingframes F14L and F15L as defined by Goebel et al., Virology (1990)179:247-266, and in the opposite orientation of ORFs F14L and F15L; thuscorresponding loci in other viruses such as poxviruses includingorthopoxviruses are included.

As used herein, attenuate toxicity of a microorganism means to reduce oreliminate deleterious or toxic effects to a host upon administration ofthe microorganism compared to the unattenuated microorganism.

As use herein, a microorganism with low toxicity means that uponadministration a microorganism does not accumulate in organs and tissuesin the host to an extent that results in damage or harm to organs orthat impact on survival of the host to a greater extent than the diseasebeing treated does.

As used herein, subject (or organism) refers to an animal, including ahuman being.

As used herein, animal includes any animal, such as, but are not limitedto primates including humans, gorillas and monkeys; rodents, such asmice and rats; fowl, such as chickens; ruminants, such as goats, cows,deer, sheep; ovine, and other animals including pigs, horses, cats,dogs, and rabbits. Non-human animals exclude humans as the contemplatedanimal.

As used herein, accumulation of a microorganism in a targeted tissuerefers to the distribution of the microorganism throughout the organismafter a time period long enough for the microbes to infect the host'sorgans or tissues. As one skilled in the art will recognize, the timeperiod for infection of a microbe will vary depending on the microbe,the targeted organ(s) or tissue(s), the immunocompetence of the host,and dosage. Generally, accumulation can be determined at time-pointsfrom about 1 day to about 1 week after infection with the microbes. Forpurposes herein, the microorganisms preferentially accumulate in thetarget tissue, such as a tumor, but are cleared from other tissues andorgans in the host to the extent that toxicity of the microorganism ismild or tolerable and at most not fatal.

As used herein, preferential accumulation refers to accumulation of amicroorganism at a first location at a higher level than accumulation ata second location. Thus, a microorganism that preferentially accumulatesin immunoprivileged tissue such as tumor relative to normal tissues ororgans refers to a microorganism that accumulates in immunoprivilegedtissue such as tumor at a higher level than the microorganismaccumulates in normal tissues or organs.

As used herein, a “compound” produced in a tumor or otherimmunoprivileged site refers to any compound that is produced in thetumor by virtue of the presence of an introduced microorganism,generally a recombinant microorganism, expressing one or more genes. Forexample, a compound produced in a tumor can be, for example, ametabolite, an encoded polypeptide or RNA, or compound that is generatedby a recombinant polypeptide (e.g., enzyme) and the cellular machineryof the tumor or immunoprivileged tissue or cells.

As used herein, a delivery vehicle for administration refers to alipid-based or other polymer-based composition, such as liposome,micelle or reverse micelle, that associates with an agent, such as amicroorganism provided herein, for delivery into a host animal.

As used herein, the term “viral vector” is used according to itsart-recognized meaning. It refers to a nucleic acid vector constructthat includes at least one element of viral origin and can be packagedinto a viral vector particle. The viral vector particles can be used forthe purpose of transferring DNA, RNA or other nucleic acids into cellseither in vitro or in vivo. Viral vectors include, but are not limitedto, retroviral vectors, vaccinia vectors, lentiviral vectors, herpesvirus vectors (e.g., HSV), baculoviral vectors, cytomegalovirus (CMV)vectors, papillomavirus vectors, simian virus (SV40) vectors, semlikiforest virus vectors, phage vectors, adenoviral vectors, andadeno-associated viral (AAV) vectors.

As used herein, oncolytic viruses refer to viruses that replicateselectively in tumor cells.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from, e.g., infection or genetic defect, andcharacterized by identifiable symptoms.

As used herein, neoplasm (neoplasia) refers to abnormal new growth, andthus means the same as tumor, which can be benign or malignant. Unlikehyperplasia, neoplastic proliferation persists even in the absence ofthe original stimulus.

As used herein, neoplastic disease refers to any disorder involvingcancer, including tumor development, growth, metastasis and progression.

As used herein, cancer is a general term for diseases caused by orcharacterized by any type of malignant tumor.

As used herein, malignant, as applies to tumors, refers to primarytumors that have the capacity of metastasis with loss of growth controland positional control.

As used herein, metastasis refers to a growth of abnormal or neoplasticcells distant from the site primarily involved by the morbid process.

As used herein, an anti-cancer agent or compound (used interchangeablywith “anti-tumor or anti-neoplastic agent”) refers to any agents orcompounds used in anti-cancer treatment. These include any agents, whenused alone or in combination with other compounds, that can alleviate,reduce, ameliorate, prevent, or place or maintain in a state ofremission of clinical symptoms or diagnostic markers associated withneoplastic disease, tumors and cancer, and can be used in methods,combinations and compositions provided herein. Exemplary anti-neoplasticagents include the microorganism provided herein used singly or incombination and/or in combination with other agents, such as alkylatingagents, antimetabolite, certain natural products, platinum coordinationcomplexes, anthracenediones, substituted ureas, methylhydrazinederivatives, adrenocortical suppressants, certain hormones, antagonistsand anti-cancer polysaccharides.

In general, for practice of the methods herein and when using themicroorganisms provided herein, the original tumor is not excised, butis employed to accumulate the administered microorganism and as thecells become leaky or lyse to become an antigen or other product factor.The antigens can serve to elicit an immune response in the host. Theantigens and products can be isolated from the tumor.

As used herein, angiogenesis is intended to encompass the totality ofprocesses directly or indirectly involved in the establishment andmaintenance of new vasculature (neovascularization), including, but notlimited to, neovascularization associated with tumors andneovascularization associated with wounds.

As used herein, by homologous means about greater than 25% nucleic acidsequence identity, such as 25%, 40%, 60%, 70%, 80%, 90% or 95%. Ifnecessary the percentage homology will be specified. The terms“homology” and “identity” are often used interchangeably but homologyfor proteins can include conservative amino acid changes. In general,sequences (protein or nucleic acid) are aligned so that the highestorder match is obtained (see, e.g.: Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; Carillo et al. (1988) SIAM J AppliedMath 48:1073). By sequence identity, the number of identical amino acidsis determined by standard alignment algorithm programs, and used withdefault gap penalties established by each supplier. Substantiallyhomologous nucleic acid molecules would hybridize typically at moderatestringency or at high stringency all along the length of the nucleicacid or along at least about 70%, 80% or 90% of the full length nucleicacid molecule of interest. Also provided are nucleic acid molecules thatcontain degenerate codons in place of codons in the hybridizing nucleicacid molecule. (For proteins, for determination of homology conservativeamino acids can be aligned as well as identical amino acids; in thiscase percentage of identity and percentage homology vary). Whether anytwo nucleic acid molecules have nucleotide sequences that are at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can be determinedusing known computer algorithms such as the “FASTA” program, using forexample, the default parameters as in Pearson et al. (1988) Proc. Natl.Acad. Sci. USA 85:2444 (other programs include the GCG program package(Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP,BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guideto Huge Computers, Mrtin J. Bishop, ed., Academic Press, San Diego,1994, and Carillo et al. (1988) SIAM J Applied Math 48:1073). Forexample, the BLAST function of the National Center for BiotechnologyInformation database can be used to determine identity. Othercommercially or publicly available programs include, DNAStar “MegAlign”program (Madison, Wis.) and the University of Wisconsin GeneticsComputer Group (UWG) “Gap” program (Madison Wis.)). Percent homology oridentity of proteins and/or nucleic acid molecules can be determined,for example, by comparing sequence information using a GAP computerprogram (e.g., Needleman et al. (1970) J. Mol. Biol. 48:443, as revisedby Smith and Waterman ((1981) Adv. Appl. Math. 2:482).

Briefly, a GAP program defines similarity as the number of alignedsymbols (i.e., nucleotides or amino acids) that are similar, divided bythe total number of symbols in the shorter of the two sequences. Defaultparameters for the GAP program can include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)and the weighted comparison matrix of Gribskov et al. (1986) Nucl. AcidsRes. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OFPROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation,pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps. Therefore, as used herein, the term “identity” represents acomparison between a test and a reference polypeptide or polynucleotide.

As used herein, recitation that amino acids of a polypeptide correspondto amino acids in a disclosed sequence, such as amino acids set forth inthe Sequence listing, refers to amino acids identified upon alignment ofthe polypeptide with the disclosed sequence to maximize identity orhomology (where conserved amino acids are aligned) using a standardalignment algorithm, such as the GAP algorithm.

As used herein, the term “at least 90% identical to” refers to percentidentities from 90 to 100% relative to the reference polypeptides.Identity at a level of 90% or more is indicative of the fact that,assuming for exemplification purposes a test and referencepolynucleotide length of 100 amino acids are compared, no more than 10%(i.e., 10 out of 100) of amino acids in the test polypeptide differsfrom that of the reference polypeptides. Similar comparisons can be madebetween a test and reference polynucleotides. Such differences can berepresented as point mutations randomly distributed over the entirelength of an amino acid sequence or they can be clustered in one or morelocations of varying length up to the maximum allowable, e.g., 10/100amino acid difference (approximately 90% identity). Differences aredefined as nucleic acid or amino acid substitutions, insertions ordeletions. At the level of homologies or identities above about 85-90%,the result should be independent of the program and gap parameters set;such high levels of identity can be assessed readily, often withoutrelying on software.

As used herein, primer refers to an oligonucleotide containing two ormore deoxyribonucleotides or ribonucleotides, typically more than three,from which synthesis of a primer extension product can be initiated.Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization and extension,such as DNA polymerase, and a suitable buffer, temperature and pH.

As used herein, chemiluminescence refers to a chemical reaction in whichenergy is specifically channeled to a molecule causing it to becomeelectronically excited and subsequently to release a photon therebyemitting visible light. Temperature does not contribute to thischanneled energy. Thus, chemiluminescence involves the direct conversionof chemical energy to light energy.

As used herein, luminescence refers to the detectable EM radiation,generally, UV, IR or visible EM radiation that is produced when theexcited product of an exergic chemical process reverts to its groundstate with the emission of light. Chemiluminescence is luminescence thatresults from a chemical reaction. Bioluminescence is chemiluminescencethat results from a chemical reaction using biological molecules (orsynthetic versions or analogs thereof) as substrates and/or enzymes.

As used herein, bioluminescence, which is a type of chemiluminescence,refers to the emission of light by biological molecules, particularlyproteins. The essential condition for bioluminescence is molecularoxygen, either bound or free in the presence of an oxygenase, aluciferase, which acts on a substrate, a luciferin. Bioluminescence isgenerated by an enzyme or other protein (luciferase) that is anoxygenase that acts on a substrate luciferin (a bioluminescencesubstrate) in the presence of molecular oxygen, and transforms thesubstrate to an excited state, which, upon return to a lower energylevel releases the energy in the form of light.

As used herein, the substrates and enzymes for producing bioluminescenceare generically referred to as luciferin and luciferase, respectively.When reference is made to a particular species thereof, for clarity,each generic term is used with the name of the organism from which itderives, for example, bacterial luciferin or firefly luciferase.

As used herein, luciferase refers to oxygenases that catalyze a lightemitting reaction. For instance, bacterial luciferases catalyze theoxidation of flavin mononucleotide (FMN) and aliphatic aldehydes, whichreaction produces light. Another class of luciferases, found amongmarine arthropods, catalyzes the oxidation of Cypridina (Vargula)luciferin, and another class of luciferases catalyzes the oxidation ofColeoptera luciferin.

Thus, luciferase refers to an enzyme or photoprotein that catalyzes abioluminescent reaction (a reaction that produces bioluminescence). Theluciferases, such as firefly and Gaussia and Renilla luciferases, areenzymes which act catalytically and are unchanged during thebioluminescence generating reaction. The luciferase photoproteins, suchas the aequorin photoprotein to which luciferin is non-covalently bound,are changed, such as by release of the luciferin, during bioluminescencegenerating reaction. The luciferase is a protein that occurs naturallyin an organism or a variant or mutant thereof, such as a variantproduced by mutagenesis that has one or more properties, such as thermalstability, that differ from the naturally-occurring protein. Luciferasesand modified mutant or variant forms thereof are well known. Forpurposes herein, reference to luciferase refers to either thephotoproteins or luciferases.

Thus, reference, for example, to “Renilla luciferase” means an enzymeisolated from member of the genus Renilla or an equivalent moleculeobtained from any other source, such as from another related copepod, orthat has been prepared synthetically. It is intended to encompassRenilla luciferases with conservative amino acid substitutions that donot substantially alter activity. Suitable conservative substitutions ofamino acids are known to those of skill in this art and can be madegenerally without altering the biological activity of the resultingmolecule. Those of skill in this art recognize that, in general, singleamino acid substitutions in non-essential regions of a polypeptide donot substantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224).

As used herein, “Aequorea GFP” refers to GFPs from the genus Aequoreaand to mutants or variants thereof. Such variants and GFPs from otherspecies are well known and are available and known to those of skill inthe art. This nomenclature encompass GFPs with conservative amino acidsubstitutions that do not substantially alter activity and physicalproperties, such as the emission spectra and ability to shift thespectral output of bioluminescence generating systems. The luciferasesand luciferin and activators thereof are referred to as bioluminescencegenerating reagents or components. Typically, a subset of these reagentswill be provided or combined with an article of manufacture.Bioluminescence will be produced upon contacting the combination withthe remaining reagents. Thus, as used herein, the component luciferases,luciferins, and other factors, such as O₂, Mg²⁺, Ca²⁺ also are referredto as bioluminescence generating reagents (or agents or components).

As used herein, bioluminescence substrate refers to the compound that isoxidized in the presence of a luciferase, and any necessary activators,and generates light. These substrates are referred to as luciferinsherein, are substrates that undergo oxidation in a bioluminescencereaction. These bioluminescence substrates include any luciferin oranalog thereof or any synthetic compound with which a luciferaseinteracts to generate light. Typical substrates include those that areoxidized in the presence of a luciferase or protein in alight-generating reaction. Bioluminescence substrates, thus, includethose compounds that those of skill in the art recognize as luciferins.Luciferins, for example, include firefly luciferin, Cypridina (alsoknown as Vargula) luciferin (coelenterazine), bacterial luciferin, aswell as synthetic analogs of these substrates or other compounds thatare oxidized in the presence of a luciferase in a reaction the producesbioluminescence.

As used herein, capable of conversion into a bioluminescence substratemeans susceptible to chemical reaction, such as oxidation or reduction,that yields a bioluminescence substrate. For example, the luminescenceproducing reaction of bioluminescent bacteria involves the reduction ofa flavin mononucleotide group (FMN) to reduced flavin mononucleotide(FMNH2) by a flavin reductase enzyme. The reduced flavin mononucleotide(substrate) then reacts with oxygen (an activator) and bacterialluciferase to form an intermediate peroxy flavin that undergoes furtherreaction, in the presence of a long-chain aldehyde, to generate light.With respect to this reaction, the reduced flavin and the long chainaldehyde are substrates.

As used herein, a bioluminescence generating system refers to the set ofreagents required to conduct a bioluminescent reaction. Thus, thespecific luciferase, luciferin and other substrates, solvents and otherreagents that can be required to complete a bioluminescent reaction froma bioluminescence system. Thus a bioluminescence generating systemrefers to any set of reagents that, under appropriate reactionconditions, yield bioluminescence. Appropriate reaction conditionsrefers to the conditions necessary for a bioluminescence reaction tooccur, such as pH, salt concentrations and temperature. In general,bioluminescence systems include a bioluminescence substrate, luciferin,a luciferase, which includes enzymes, luciferases and photoproteins, andone or more activators. A specific bioluminescence system may beidentified by reference to the specific organism from which theluciferase derives; for example, the Renilla bioluminescence systemincludes a Renilla luciferase, such as a luciferase isolated from theRenilla or produced using recombinant means or modifications of theseluciferases. This system also includes the particular activatorsnecessary to complete the bioluminescence reaction, such as oxygen and asubstrate with which the luciferase reacts in the presence of the oxygento produce light.

As used herein, a fluorescent protein refers to a protein that possessesthe ability to fluoresce (i.e., to absorb energy at one wavelength andemit it at another wavelength). For example, a green fluorescent proteinrefers to a polypeptide that has a peak in the emission spectrum atabout 510 nm.

As used herein, genetic therapy or gene therapy involves the transfer ofheterologous nucleic acid, such as DNA, into certain cells, targetcells, of a mammal, particularly a human, with a disorder or conditionsfor which such therapy is sought. The nucleic acid, such as DNA, isintroduced into the selected target cells, such as directly or in avector or other delivery vehicle, in a manner such that the heterologousnucleic acid, such as DNA, is expressed and a therapeutic productencoded thereby is produced. Alternatively, the heterologous nucleicacid, such as DNA, can in some manner mediate expression of DNA thatencodes the therapeutic product, or it can encode a product, such as apeptide or RNA that in some manner mediates, directly or indirectly,expression of a therapeutic product. Genetic therapy also can be used todeliver nucleic acid encoding a gene product that replaces a defectivegene or supplements a gene product produced by the mammal or the cell inwhich it is introduced. The introduced nucleic acid can encode atherapeutic compound, such as a growth factor inhibitor thereof, or atumor necrosis factor or inhibitor thereof, such as a receptor therefor,that is not normally produced in the mammalian host or that is notproduced in therapeutically effective amounts or at a therapeuticallyuseful time. The heterologous nucleic acid, such as DNA, encoding thetherapeutic product can be modified prior to introduction into the cellsof the afflicted host in order to enhance or otherwise alter the productor expression thereof. Genetic therapy also can involve delivery of aninhibitor or repressor or other modulator of gene expression.

As used herein, heterologous nucleic acid is nucleic acid that is notnormally produced in vivo by the microorganism from which it isexpressed or that is produced by a microorganism but is at a differentlocus or expressed differently or that mediates or encodes mediatorsthat alter expression of endogenous nucleic acid, such as DNA, byaffecting transcription, translation, or other regulatable biochemicalprocesses. Heterologous nucleic acid is often not endogenous to the cellinto which it is introduced, but has been obtained from another cell orprepared synthetically. Heterologous nucleic acid, however, can beendogenous, but is nucleic acid that is expressed from a different locusor altered in its expression or sequence. Generally, although notnecessarily, such nucleic acid encodes RNA and proteins that are notnormally produced by the cell or in the same way in the cell in which itis expressed. Heterologous nucleic acid, such as DNA, also can bereferred to as foreign nucleic acid, such as DNA. Thus, heterologousnucleic acid or foreign nucleic acid includes a nucleic acid moleculenot present in the exact orientation or position as the counterpartnucleic acid molecule, such as DNA, is found in a genome. It also canrefer to a nucleic acid molecule from another organism or species (i.e.,exogenous). Any nucleic acid, such as DNA, that one of skill in the artwould recognize or consider as heterologous or foreign to the cell inwhich the nucleic acid is expressed is herein encompassed byheterologous nucleic acid; heterologous nucleic acid includesexogenously added nucleic acid that also is expressed endogenously.Examples of heterologous nucleic acid include, but are not limited to,nucleic acid that encodes traceable marker proteins, such as a proteinthat confers drug resistance, nucleic acid that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and nucleic acid, such as DNA, that encodes other types of proteins,such as antibodies. Antibodies that are encoded by heterologous nucleicacid can be secreted or expressed on the surface of the cell in whichthe heterologous nucleic acid has been introduced.

As used herein, a therapeutically effective product for gene therapy isa product that is encoded by heterologous nucleic acid, typically DNA,(or an RNA product such as dsRNA, RNAi, including siRNA, that, uponintroduction of the nucleic acid into a host, a product is expressedthat ameliorates or eliminates the symptoms, manifestations of aninherited or acquired disease or that cures the disease. Also includedare biologically active nucleic acid molecules, such as RNAi andantisense.

As used herein, cancer or tumor treatment or agent refers to anytherapeutic regimen and/or compound that, when used alone or incombination with other treatments or compounds, can alleviate, reduce,ameliorate, prevent, or place or maintain in a state of remission ofclinical symptoms or diagnostic markers associated with deficientangiogenesis.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, which are optionally labeled, such as with a detectable label,such as a fluorescent or radiolabel, single-stranded molecules areprovided. Such molecules are typically of a length such that theirtarget is statistically unique or of low copy number (typically lessthan 5, generally less than 3) for probing or priming a library.Generally a probe or primer contains at least 14, 16 or 30 contiguousnucleotides of sequence complementary to or identical to a gene ofinterest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleicacids long.

As used herein, operative linkage of heterologous nucleic acids toregulatory and effector sequences of nucleotides, such as promoters,enhancers, transcriptional and translational stop sites, and othersignal sequences refers to the relationship between such nucleic acid,such as DNA, and such sequences of nucleotides. For example, operativelinkage of heterologous DNA to a promoter refers to the physicalrelationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. Thus, operatively linked or operationally associated refers to thefunctional relationship of nucleic acid, such as DNA, with regulatoryand effector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences. For example, operative linkage of DNA to a promoter refers tothe physical and functional relationship between the DNA and thepromoter such that the transcription of such DNA is initiated from thepromoter by an RNA polymerase that specifically recognizes, binds to andtranscribes the DNA. In order to optimize expression and/or in vitrotranscription, it can be necessary to remove, add or alter 5′untranslated portions of the clones to eliminate extra, potentiallyinappropriate alternative translation initiation start) codons or othersequences that can interfere with or reduce expression, either at thelevel of transcription or translation. Alternatively, consensus ribosomebinding sites (see, e.g., Kozak J. Biol. Chem. 266:19867-19870 (1991))can be inserted immediately 5′ of the start codon and can enhanceexpression. The desirability of (or need for) such modification can beempirically determined.

As used herein, a sequence complementary to at least a portion of anRNA, with reference to antisense oligonucleotides, means a sequence ofnucleotides having sufficient complementarity to be able to hybridizewith the RNA, generally under moderate or high stringency conditions,forming a stable duplex; in the case of double-stranded antisensenucleic acids, a single strand of the duplex DNA (or dsRNA) can thus betested, or triplex formation can be assayed. The ability to hybridizedepends on the degree of complementarity and the length of the antisensenucleic acid. Generally, the longer the hybridizing nucleic acid, themore base mismatches with an encoding RNA it can contain and still forma stable duplex (or triplex, as the case can be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

As used herein, amelioration of the symptoms of a particular disordersuch as by administration of a particular pharmaceutical composition,refers to any lessening, whether permanent or temporary, lasting ortransient that can be attributed to or associated with administration ofthe composition.

As used herein, antisense polynucleotides refer to synthetic sequencesof nucleotide bases complementary to mRNA or the sense strand ofdouble-stranded DNA. A mixture of sense and antisense polynucleotidesunder appropriate conditions leads to the binding of the two molecules,or hybridization. When these polynucleotides bind to (hybridize with)mRNA, inhibition of protein synthesis (translation) occurs. When thesepolynucleotides bind to double-stranded DNA, inhibition of RNA synthesis(transcription) occurs. The resulting inhibition of translation and/ortranscription leads to an inhibition of the synthesis of the proteinencoded by the sense strand. Antisense nucleic acid molecules typicallycontain a sufficient number of nucleotides to specifically bind to atarget nucleic acid, generally at least 5 contiguous nucleotides, oftenat least 14 or 16 or 30 contiguous nucleotides or modified nucleotidescomplementary to the coding portion of a nucleic acid molecule thatencodes a gene of interest.

As used herein, antibody refers to an immunoglobulin, whether natural orpartially or wholly synthetically produced, including any derivativethereof that retains the specific binding ability of the antibody. Henceantibody includes any protein having a binding domain that is homologousor substantially homologous to an immunoglobulin binding domain.Antibodies include members of any immunoglobulin class, including IgG,IgM, IgA, IgD and IgE.

As used herein, antibody fragment refers to any derivative of anantibody that is less then full length, retaining at least a portion ofthe full-length antibody's specific binding ability. Examples ofantibody fragments include, but are not limited to, Fab, Fab′, F(ab)2,single-chain Fvs (scFV), FV, dsFV diabody and Fd fragments. The fragmentcan include multiple chains linked together, such as by disulfidebridges. An antibody fragment generally contains at least about 50 aminoacids and typically at least 200 amino acids.

As used herein, a Fv antibody fragment is composed of one variable heavychain domain (V_(H)) and one variable light chain domain linked bynoncovalent interactions.

As used herein, a dsFV refers to an Fv with an engineered intermoleculardisulfide bond, which stabilizes the V_(H)-V_(L) pair.

As used herein, a F(ab)2 fragment is an antibody fragment that resultsfrom digestion of an immunoglobulin with pepsin at pH 4.0-4.5; it can berecombinantly produced to produce the equivalent fragment.

As used herein, Fab fragments are antibody fragments that result fromdigestion of an immunoglobulin with papain; it can be recombinantlyproduced to produce the equivalent fragment.

As used herein, scFVs refer to antibody fragments that contain avariable light chain (V_(L)) and variable heavy chain (V_(H)) covalentlyconnected by a polypeptide linker in any order. The linker is of alength such that the two variable domains are bridged withoutsubstantial interference. Included linkers are (Gly-Ser)n residues withsome Glu or Lys residues dispersed throughout to increase solubility.

As used herein, humanized antibodies refer to antibodies that aremodified to include human sequences of amino acids so thatadministration to a human does not provoke an immune response. Methodsfor preparation of such antibodies are known. For example, to producesuch antibodies, the encoding nucleic acid in the hybridoma or otherprokaryotic or eukaryotic cell, such as an E. coli or a CHO cell, thatexpresses the monoclonal antibody is altered by recombinant nucleic acidtechniques to express an antibody in which the amino acid composition ofthe non-variable region is based on human antibodies. Computer programshave been designed to identify such non-variable regions.

As used herein, diabodies are dimeric scFV; diabodies typically haveshorter peptide linkers than scFvs, and they generally dimerize.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein the term assessing or determining is intended to includequantitative and qualitative determination in the sense of obtaining anabsolute value for the activity of a product, and also of obtaining anindex, ratio, percentage, visual or other value indicative of the levelof the activity. Assessment can be direct or indirect.

As used herein, biological activity refers to the in vivo activities ofa compound or microorganisms or physiological responses that result uponin vivo administration thereof or of composition or other mixture.Biological activity, thus, encompasses therapeutic effects andpharmaceutical activity of such compounds, compositions and mixtures.Biological activities can be observed in in vitro systems designed totest or use such activities.

As used herein, an effective amount of a microorganism or compound fortreating a particular disease is an amount that is sufficient toameliorate, or in some manner reduce the symptoms associated with thedisease. Such an amount can be administered as a single dosage or can beadministered according to a regimen, whereby it is effective. The amountcan cure the disease but, typically, is administered in order toameliorate the symptoms of the disease. Repeated administration can berequired to achieve the desired amelioration of symptoms.

As used herein equivalent, when referring to two sequences of nucleicacids, means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides or other molecules, it means thatthe two proteins or peptides have substantially the same amino acidsequence with only amino acid substitutions (such as, but not limitedto, conservative changes) or structure and the any changes do notsubstantially alter the activity or function of the protein or peptide.When equivalent refers to a property, the property does not need to bepresent to the same extent (e.g., two peptides can exhibit differentrates of the same type of enzymatic activity), but the activities areusually substantially the same. Complementary, when referring to twonucleotide sequences, means that the two sequences of nucleotides arecapable of hybridizing, typically with less than 25%, 15% or 5%mismatches between opposed nucleotides. If necessary, the percentage ofcomplementarity will be specified. Typically the two molecules areselected such that they will hybridize under conditions of highstringency.

As used herein, an agent or compound that modulates the activity of aprotein or expression of a gene or nucleic acid either decreases orincreases or otherwise alters the activity of the protein or, in somemanner, up- or down-regulates or otherwise alters expression of thenucleic acid in a cell.

As used herein, a method for treating or preventing neoplastic diseasemeans that any of the symptoms, such as the tumor, metastasis thereof,the vascularization of the tumors or other parameters by which thedisease is characterized are reduced, ameliorated, prevented, placed ina state of remission, or maintained in a state of remission. It alsomeans that the hallmarks of neoplastic disease and metastasis can beeliminated, reduced or prevented by the treatment. Non-limiting examplesof the hallmarks include uncontrolled degradation of the basementmembrane and proximal extracellular matrix, migration, division, andorganization of the endothelial cells into new functioning capillaries,and the persistence of such functioning capillaries.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound is regenerated by metabolicprocesses. The prodrug can be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388-392).

As used herein, a promoter region or promoter element or regulatoryregion refers to a segment of DNA or RNA that controls transcription ofthe DNA or RNA to which it is operatively linked. The promoter regionincludes specific sequences that are sufficient for RNA polymeraserecognition, binding and transcription initiation. This portion of thepromoter region is referred to as the promoter. In addition, thepromoter region includes sequences that modulate this recognition,binding and transcription initiation activity of RNA polymerase. Thesesequences can be cis acting or can be responsive to trans actingfactors. Promoters, depending upon the nature of the regulation, can beconstitutive or regulated. Exemplary promoters contemplated for use inprokaryotes include the bacteriophage T7 and T3 promoters.

As used herein, a receptor refers to a molecule that has an affinity fora ligand. Receptors can be naturally-occurring or synthetic molecules.Receptors also can be referred to in the art as anti-ligands. As usedherein, the receptor and anti-ligand are interchangeable. Receptors canbe used in their unaltered state or bound to other polypeptides,including as homodimers. Receptors can be attached to, covalently ornoncovalently, or in physical contact with, a binding member, eitherdirectly or indirectly via a specific binding substance or linker.Examples of receptors, include, but are not limited to: antibodies, cellmembrane receptors surface receptors and internalizing receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells, or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles.

As used herein, sample refers to anything that can contain an analytefor which an analyte assay is desired. The sample can be a biologicalsample, such as a biological fluid or a biological tissue. Examples ofbiological fluids include urine, blood, plasma, serum, saliva, semen,stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid orthe like. Biological tissues are aggregates of cells, usually of aparticular kind together with their intercellular substance that formone of the structural materials of a human, animal, plant, bacterial,fungal or viral structure, including connective, epithelium, muscle andnerve tissues. Examples of biological tissues also include organs,tumors, lymph nodes, arteries and individual cell(s).

As used herein: stringency of hybridization in determining percentagemismatch is as follows:

1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.

2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.

3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.

Those of skill in this art know that the washing step selects for stablehybrids and also know the ingredients of SSPE (see, e.g., Sambrook, E.F. Fritsch, T. Maniatis, in: Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989), vol. 3, p. B.13, see, also,numerous catalogs that describe commonly used laboratory solutions).SSPE is pH 7.4 phosphate-buffered 0.18 M NaCl. Further, those of skillin the art recognize that the stability of hybrids is determined by Tm,which is a function of the sodium ion concentration and temperature:(Tm=81.5° C.-16.6(log 10[Na+])+0.41(% G+C)−600/1)), so that the onlyparameters in the wash conditions critical to hybrid stability aresodium ion concentration in the SSPE (or SSC) and temperature. Anynucleic acid molecules provided herein can also include those thathybridize under conditions of at least low stringency, generallymoderate or high stringency, along at least 70, 80, 90% of the fulllength of the disclosed molecule. It is understood that equivalentstringencies can be achieved using alternative buffers, salts andtemperatures. By way of example and not limitation, procedures usingconditions of low stringency are as follows (see also Shilo andWeinberg, Proc. Natl. Acad. Sci. USA 78:6789-6792 (1981)):

Filters containing DNA are pretreated for 6 hours at 40° C. in asolution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mMEDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 gg/ml denatured salmonsperm DNA (10×SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate,adjusted to a pH of 7). Hybridizations are carried out in the samesolution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2%BSA, 100 Tg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and5-20×10⁶ cpm ³²P-labeled probe is used. Filters are incubated inhybridization mixture for 18-20 hours at 40° C., and then washed for 1.5hours at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4),5 mM EDTA, and 0.1% SDS. The wash solution is replaced with freshsolution and incubated an additional 1.5 hours at 60° C. Filters areblotted dry and exposed for autoradiography. If necessary, filters arewashed for a third time at 65-68° C. and reexposed to film. Otherconditions of low stringency which can be used are well known in the art(e.g., as employed for cross-species hybridizations).

By way of example and not way of limitation, procedures using conditionsof moderate stringency include, for example, but are not limited to,procedures using such conditions of moderate stringency are as follows:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6×SSC, 5×Denhart's solution, 0.5% SDS and 100 μg/mldenatured salmon sperm DNA. Hybridizations are carried out in the samesolution and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 hours at 55° C., and thenwashed twice for 30 minutes at 60° C. in a solution containing 1×SSC and0.1% SDS. Filters are blotted dry and exposed for autoradiography. Otherconditions of moderate stringency which can be used are well-known inthe art. Washing of filters is done at 37° C. for 1 hour in a solutioncontaining 2×SSC, 0.1% SDS. By way of example and not way of limitation,procedures using conditions of high stringency are as follows:Prehybridization of filters containing DNA is carried out for 8 hours toovernight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 Tg/mldenatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C. in prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters isdone at 37° C. for 1 hour in a solution containing 2×SSC, 0.01% PVP,0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at50° C. for 45 minutes before autoradiography. Other conditions of highstringency which can be used are well known in the art.

The term substantially identical or homologous or similar varies withthe context as understood by those skilled in the relevant art andgenerally means at least 60% or 70%, preferably means at least 80%, morepreferably at least 90%, and most preferably at least 95%, 96%, 97%,98%, 99% or greater identity.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound can, however, be amixture of stereoisomers or isomers. In such instances, furtherpurification might increase the specific activity of the compound.

As used herein, a molecule, such as an antibody, that specifically bindsto a polypeptide typically has a binding affinity (Ka) of at least about10⁶ l/mol, 10⁷ l/mol, 10⁸ l/mol, 10⁹ l/mol, 10¹⁰ l/mol or greater andbinds to a protein of interest generally with at least 2-fold, 5-fold,generally 10-fold or even 100-fold or greater, affinity than to otherproteins. For example, an antibody that specifically binds to theprotease domain compared to the full-length molecule, such as thezymogen form, binds with at least about 2-fold, typically 5-fold or10-fold higher affinity, to a polypeptide that contains only theprotease domain than to the zymogen form of the full-length. Suchspecific binding also is referred to as selective binding. Thus,specific or selective binding refers to greater binding affinity(generally at least 2-fold, 5-fold, 10-fold or more) to a targeted siteor locus compared to a non-targeted site or locus.

As used herein, the terms a therapeutic agent, therapeutic compound,therapeutic regimen, or chemotherapeutic include conventional drugs anddrug therapies, including vaccines, which are known to those skilled inthe art.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of themicroorganisms described and provided herein.

As used herein, proliferative disorders include any disorders involvingabnormal proliferation of cells. Such disorders include, but are notlimited to, neoplastic diseases, psoriasis, restenosis, maculardegeneration, diabetic retinopathies, inflammatory responses anddisorders, including wound healing responses.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vectorsare well known to those of skill in the art. An expression vectorincludes vectors capable of expressing DNA that is operatively linkedwith regulatory sequences, such as promoter regions, that are capable ofeffecting expression of such DNA fragments. Thus, an expression vectorrefers to a recombinant DNA or RNA construct, such as a plasmid, aphage, recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, a combination refers to any association between two oramong more items.

As used herein, a composition refers to any mixture. It can be asolution, a suspension, an emulsion, liquid, powder, a paste, aqueous,non-aqueous or any combination thereof.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a kit is a packaged combination optionally includinginstructions for use of the combination and/or other reactions andcomponents for such use.

For clarity of disclosure, and not by way of limitation, the detaileddescription is divided into the subsections that follow.

B. MICROORGANISMS FOR TUMOR-SPECIFIC THERAPY

Provided herein are microorganisms, and methods for making and usingsuch microorganisms for therapy of neoplastic disease and otherproliferative disorders and inflammatory disorders. The microbe (ormicroorganism)-mediated treatment methods provided herein involveadministration of microorganisms to hosts, accumulation of themicroorganism in the targeted cell or tissue, such as in a tumor,resulting in leaking or lysing of the cells, whereby an immune responseagainst leaked or released antigens is mounted, thereby resulting in aninhibition of the tissues or cells in which the microorganismaccumulates.

In addition to the gene therapeutic methods of cancer treatment, liveattenuated microorganisms can be used for vaccination, such as in cancervaccination or antitumor immunity. Immunization, for example, against atumor can include a tumor-specific T-cell-mediated response throughmicrobe-delivered antigens or cytokines. To do so, the microbes can bespecifically targeted to the tumor tissues, with minimal infection toany other key organs and also can be modified or provided to produce theantigens and/or cytokines.

The microorganisms provided herein and the use of such microorganismsherein can accumulate in immunoprivileged cells or immunoprivilegedtissues, including tumors and/or metastases, and also including woundedtissues and cells. While the microorganisms provided herein cantypically be cleared from the subject to whom the microorganisms areadministered by activity of the subject's immune system, microorganismscan nevertheless accumulate, survive and proliferate in immunoprivilegedcells and tissues such as tumors because such immunoprivileged areas aresequestered from the host's immune system. Accordingly, the methodsprovided herein, as applied to tumors and/or metastases, and therapeuticmethods relating thereto, can readily be applied to otherimmunoprivileged cells and tissues, including wounded cells and tissues.

1. Characteristics

The microorganisms provided herein and used in the methods herein areattenuated, immunogenic, and replication competent.

a. Attenuated

The microbes used in the methods provided herein are typicallyattenuated. Attenuated microbes have a decreased capacity to causedisease in a host. The decreased capacity can result from any of avariety of different modifications to the ability of a microbe to bepathogenic. For example, a microbe can have reduced toxicity, reducedability to accumulate in non-tumorous organs or tissue, reduced abilityto cause cell lysis or cell death, or reduced ability to replicatecompared to the non-attenuated form thereof. The attenuated microbesprovided herein, however, retain at least some capacity to replicate andto cause immunoprivileged cells and tissues, such as tumor cells to leakor lyse, undergo cell death, or otherwise cause or enhance an immuneresponse to immunoprivileged cells and tissues, such as tumor cells.

i. Reduced Toxicity

Microbes can be toxic to their hosts by manufacturing one or morecompounds that worsen the health condition of the host. Toxicity to thehost can be manifested in any of a variety of manners, including septicshock, neurological effects, or muscular effects. The microbes providedherein can have a reduced toxicity to the host. The reduced toxicity ofa microbe of the present methods and compositions can range from atoxicity in which the host experiences no toxic effects, to a toxicityin which the host does not typically die from the toxic effects of themicrobes. In some embodiments, the microbes are of a reduced toxicitysuch that a host typically has no significant long-term effect from thepresence of the microbes in the host, beyond any effect on tumorous,metastatic or necrotic organs or tissues. For example, the reducedtoxicity can be a minor fever or minor infection, which lasts for lessthan about a month, and following the fever or infection, the hostexperiences no adverse effects resultant from the fever or infection. Inanother example, the reduced toxicity can be measured as anunintentional decline in body weight of about 5% or less for the hostafter administration of the microbes. In other examples, the microbe hasno toxicity to the host.

Exemplary vaccinia viruses of the LIVP strain (a widely availableattenuated Lister strain) that have reduced toxicity compared to othervaccinia viruses employed and are further modified. Modified LIVP wereprepared. These modified LIVP include insertions in the TK and/or HAgenes and in the locus designed F3. As an example of reduced toxicity,recombinant vaccinia viruses were tested for their toxicity to mice withimpaired immune systems (nude mice) relative to the corresponding wildtype vaccinia virus. Intravenous (i.v.) injection of wild type vacciniavirus VGL (strain LIVP) at 1×10⁷ PFU/mouse causes toxicity in nude mice:three mice out of seven lost the weight and died (one mouse died in oneweek after virus injection, one mouse died ten days after virusinjection. Similar modifications can be made to other pox viruses andother viruses to reduce toxicity thereof. Such modifications can beempirically identified, if necessary.

ii. Accumulate in Immunoprivileged Cells and Tissues, Such as Tumor, notSubstantially in Other Organs

Microbes can accumulate in any of a variety of tissues and organs of thehost. Accumulation can be evenly distributed over the entire hostorganism, or can be concentrated in one or a few organs or tissues, Themicrobes provided herein can accumulate in targeted tissues, such asimmunoprivileged cells and tissues, such as tumors and also metastases.In some embodiments, the microbes provided herein exhibit accumulationin immunoprivileged cells and tissues, such as tumor cells relative tonormal organs or tissues that is equal to or greater than theaccumulation that occurs with wild type microbes. In other embodimentsthe microbes provided herein exhibit accumulation in immunoprivilegedcells and tissues, such as tumor cells that is equal to or greater thanthe accumulation in any other particular organ or tissue. For example,the microbes provided herein can demonstrate an accumulation inimmunoprivileged cells and tissues, such as tumor cells that is at leastabout 2-fold greater, at least about 5-fold greater, at least about10-fold greater, at least about 100-fold greater, at least about1,000-fold greater, at least about 10,000-fold greater, at least about100,000-fold greater, or at least about 1,000,000-fold greater, than theaccumulation in any other particular organ or tissue.

In some embodiments, a microbe can accumulate in targeted tissues andcells, such as immunoprivileged cells and tissues, such as tumor cells,without accumulating in one or more selected tissues or organs. Forexample, a microbe can accumulate in tumor cells without accumulating inthe brain. In another example, a microbe can accumulate in tumor cellswithout accumulating in neural cells. In another example, a microbe canaccumulate in tumor cells without accumulating in ovaries. In anotherexample, a microbe can accumulate in tumor cells without accumulating inthe blood. In another example, a microbe can accumulate in tumor cellswithout accumulating in the heart. In another example, a microbe canaccumulate in tumor cells without accumulating in the bladder. Inanother example, a microbe can accumulate in tumor cells withoutaccumulating in testes. In another example, a microbe can accumulate intumor cells without accumulating in the spleen. In another example, amicrobe can accumulate in tumor cells without accumulating in the lungs.

One skilled in the art can determine the desired capability for themicrobes to selectively accumulate in targeted tissue or cells, such asin an immunoprivileged cells and tissues, such as tumor rather thannon-target organs or tissues, according to a variety of factors known inthe art, including, but not limited to, toxicity of the microbes,dosage, tumor to be treated, immunocompetence of host, and disease stateof the host.

Provided herein as an example of selective accumulation inimmunoprivileged cells and tissues, such as tumors relative to normalorgans or tissues, presence of various vaccinia viruses was assayed intumor samples and different organs. Wild type VGL virus (LIVP) wasrecovered from tumor, testes, bladder, and liver and as well as frombrain. Recombinant virus RVGL9 was found mostly in tumors, and no viruswas recovered from brain tissue in six tested animals. Therefore, thisfinding demonstrates the tumor accumulation properties of a recombinantvaccinia virus of the LIVP strain with an insertion in the F3 gene fortumor therapy purposes.

iii. Ability to Elicit or Enhance Immune Response to Tumor Cells

The microorganisms herein cause or enhance an immune response toantigens in the targeted tissues or cells, such as immunoprivilegedcells and tissues, such as tumor cells. The immune response can betriggered by any of a variety of mechanisms, including the presence ofimmunostimulatory cytokines and the release of antigenic compounds thatcan cause an immune response.

Cells, in response to an infection such as a microorganismal infection,can send out signals to stimulate an immune response against the cells.Exemplary signals sent from such cells include antigens, cytokines andchemokines such as interferon-gamma and interleukin-15. Themicroorganism provided herein can cause targeted cells to send out suchsignals in response to infection by the microbes, resulting in astimulation of the host's immune system against the targeted cells ortissues, such as tumor cells.

In another embodiment, targeted cells or tissues, such as tumor cells,can contain one or more compounds that can be recognized by the host'simmune system in mounting an immune response against a tumor. Suchantigenic compounds can be compounds on the cell surface or the tumorcell, and can be protein, carbohydrate, lipid, nucleic acid, orcombinations thereof. Microbe-mediated release of antigenic compoundscan result in triggering the host's immune system to mount an immuneresponse against the tumor. The amount of antigenic compound released bythe tumor cells is any amount sufficient to trigger an immune responsein a subject; for example, the antigenic compounds released from one ormore tumor cells can trigger a host immune response in the organism thatis known to be accessible to leukocytes.

The time duration of antigen release is an amount of time sufficient forthe host to establish an immune response to one or more tumor antigens.In some embodiments, the duration is an amount of time sufficient forthe host to establish a sustained immune response to one or more tumorantigens. One skilled in the art can determine such a time durationbased on a variety of factors affecting the time duration for a subjectto develop an immune response, including the level of the tumor antigenin the subject, the number of different tumor antigens, the antigenicityof the antigen, the immunocompetence of the host, and the access of theantigenic material to the vasculature of the host. Typically, theduration of antigen release can be at least about a week, at least about10 days, at least about two weeks, or at least about a month.

The microorganism provided herein can have any of a variety ofproperties that can cause target cells and tissues, such as tumor cells,to release antigenic compounds. Exemplary properties are the ability tolyse cells and the ability to elicit apoptosis in tumor cells. Microbesthat are unable to lyse tumor cells or cause tumor cell death cannevertheless be used in the methods provided herein when such microbescan cause some release or display of antigenic compounds from tumorcells. A variety of mechanisms for antigen release or display withoutlysis or cell death are known in the art, and any such mechanism can beused by the microbes provided herein, including, but not limited to,secretion of antigenic compounds, enhanced cell membrane permeability,or altered cell surface expression or altered MHC presentation in tumorcells when the tumor cells can be accessed by the host's immune system.Regardless of the mechanism by which the host's immune system isactivated, the net result of the presence of the microbes in the tumoris a stimulation of the host's immune system, at least in part, againstthe tumor cells. In one example, the microbes can cause an immuneresponse against tumor cells not infected by the microbes.

In one embodiment, the microbes provided herein can cause tumor cells torelease an antigen that is not present on the tumor cell surface. Tumorcells can produce compounds such as proteins that can cause an immuneresponse; however, in circumstances in which the antigenic compound isnot on the tumor cell surface, the tumor can proliferate, and evenmetastasize, without the antigenic compound causing an immune response.Within the scope of the present methods, the microbes provided hereincan cause antigenic compounds within the cell to release away from thecell and away from the tumor, which can result in triggering an immuneresponse to such an antigen. Even if not all cells of a tumor arereleasing antigens, the immune response can initially be targeted towardthe “leaky” tumor cells, and the bystander effect of the immune responsecan result in further tumor cell death around the “leaky” tumor cells.

iv. Balance of Pathogenicity and Release of Tumor Antigens

Typical methods of involving treatment of targeted cells and tissues,such as immunoprivileged cells and tissues, such as tumors, are designedto cause rapid and complete removal thereof. For example, many viruses,bacterial or eukaryotic cells can cause lysis and/or apoptosis in avariety of cells, including tumor cells. Microorganisms that canvigorously lyse or cause cell death can be highly pathogenic, and caneven kill the host. Furthermore, therapeutic methods based upon suchrapid and complete lysis are typically therapeutically ineffective.

In contrast, the microorganisms provided herein are not aggressive incausing cell death or lysis. They can have only a limited or no abilityto cause cell death as long as they accumulate in the target cells ortissues and result in alteration of cell membranes to cause leakage ofantigens against which an immune response is mounted. It is desirablethat their apoptotic or lytic effect is sufficiently slow or ineffectiveto permit sufficient antigenic leakage for a sufficient time for thehost to mount an effective immune response against the target tissues.Such immune response alone or in combination with the lytic/apoptoticeffect of the microorganism results in elimination of the target tissueand also elimination of future development, such as metastases andreoccurrence, of such tissues or cells. While the microbes providedherein can have a limited ability to cause cell death, the microbesprovided herein can nevertheless stimulate the host's immune system toattack tumor cells. As a result, such microorganisms also are typicallyunlikely to have substantial toxicity to the host.

In one embodiment, the microbes have a limited, or no, ability to causetumor cell death, while still causing or enhancing an immune responseagainst tumor cells. In one example, the rate of microorganism-mediatedtumor cell death is less than the rate of tumor cell growth orreplication. In another example, the rate of microorganism-mediatedtumor cell death is slow enough for the host to establish a sustainedimmune response to one or more tumor antigens. Typically, the time forof cell death is sufficient to establish an anti-tumor immune responseand can be at least about a week, at least about 10 days, at least abouttwo weeks, or at least about a month, depending upon the host and thetargeted cells or tissues.

In another embodiment, the microbes provided herein can cause cell deathin tumor cells, without causing substantial cell death in non-tumortissues. In such an embodiment, the microbes can aggressively kill tumorcells, as long as no substantial cell death occurs in non-tumor cells,and optionally, so long as the host has sufficient capability to mountan immune response against the tumor cells.

In one embodiment, the ability of the microbes to cause cell death isslower than the host's immune response against the microbes. The abilityfor the host to control infection by the microbes can be determined bythe immune response (e.g., antibody titer) against microorganismalantigens. Typically, after the host has mounted immune response againstthe microbes, the microbes can have reduced pathogenicity in the host.Thus, when the ability of the microbes to cause cell death is slowerthan the host's immune response against the microbes, microbe-mediatedcell death can occur without risk of serious disease or death to thehost. In one example, the ability of the microbes to cause tumor celldeath is slower than the host's immune response against the microbes.

b. Immunogenicity

The microorganisms provided herein also can be immunogenic. Animmunogenic microorganism can create a host immune response against themicroorganism. In one embodiment, the microorganisms can be sufficientlyimmunogenic to result in a large anti-(microorganism) antibody titer.The microorganisms provided herein can have the ability to elicit animmune response. The immune response can be activated in response toviral antigens or can be activated as a result ofmicroorganismal-infection induced cytokine or chemokine production.Immune response against the microorganism can decrease the likelihood ofpathogenicity toward the host organism.

Immune response against the microorganism also can result in targettissue or cell, such as tumor cell, killing. In one embodiment, theimmune response against microorganismal infection can result in animmune response against tumor cells, including developing antibodiesagainst tumor antigens. In one example, an immune response mountedagainst the microorganism can result in tumor cell killing by the“bystander effect,” where uninfected tumor cells nearby infected tumorcells are killed at the same time as infected cells, or alternatively,where uninfected tumor cells nearby extracellular microorganisms arekilled at the same time as the microorganisms. As a result of bystandereffect tumor cell death, tumor cell antigens can be released from cells,and the host organism's immune system can mount an immune responseagainst tumor cell antigens, resulting in an immune response against thetumor itself.

In one embodiment, the microorganism can be selected or modified toexpress one or more antigenic compounds, including superantigeniccompounds. The antigenic compounds such as superantigens can beendogenous gene products or can be exogenous gene products.Superantigens, including toxoids, are known in the art and describedelsewhere herein.

c. Replication Competent

The microorganisms provided herein can be replication competent. In avariety of viral or bacterial systems, the administered microorganism isrendered replication incompetent to limit pathogenicity risk to thehost. While replication incompetence can protect the host from themicroorganism, that also limits the ability of the microorganism toinfect and kill tumor cells, and typically results in only a short-livedeffect. In contrast, the microorganisms provided herein can beattenuated but replication competent, resulting in low toxicity to thehost and accumulation mainly or solely in tumors. Thus, themicroorganisms provided herein can be replication competent withoutcreating a pathogenicity risk to the host.

Attenuation of the microorganisms provided herein can include, but isnot limited to, reducing the replication competence of themicroorganism. For example, a microorganism can be modified to decreaseor eliminate an activity related to replication, such as atranscriptional activator that regulates replication in themicroorganism. In an example, a microorganism, such as a virus, can havethe viral thymidine kinase gene modified.

d. Genetic Variants

The microorganisms provided herein can be modified from their wild typeform. Modifications can include any of a variety of changes, andtypically include changes to the genome or nucleic acid molecules of themicroorganisms. Exemplary nucleic acid molecular modifications includetruncations, insertions, deletions and mutations. In an exemplarymodification, a microorganismal gene can be modified by truncation,insertion, deletion or mutation. In an exemplary insertion, an exogenousgene can be inserted into the genome of the microorganism.

i. Modified Characteristics

Modifications of the microorganisms provided herein can result in amodification of microorganismal characteristics, including thoseprovided herein such as pathogenicity, toxicity, ability topreferentially accumulate in tumor, ability to lyse cells or cause celldeath, ability to elicit an immune response against tumor cells,immunogenicity, replication competence. Variants can be obtained bygeneral methods such as mutagenesis and passage in cell or tissueculture and selection of desired properties, as is known in the art, asexemplified for respiratory syncytial virus in Murphy et al., Virus Res.1994, 32:13-26.

Variants also can be obtained by mutagenic methods in which nucleic acidresidues of the microorganism are added, removed or modified relative tothe wild type. Any of a variety of known mutagenic methods can be used,including recombination-based methods, restriction endonuclease-basedmethods, and PCR-based methods. Mutagenic methods can be directedagainst particular nucleotide sequences such as genes, or can be random,where selection methods based on desired characteristics can be used toselect mutated microorganisms. Any of a variety of microorganismalmodifications can be made, according to the selected microorganism andthe particular known modifications of the selected microorganism.

ii. Exogenous Gene Expression

The microorganisms provided herein also can have the ability to expressone or more exogenous genes. Gene expression can include expression of aprotein encoded by a gene and/or expression of an RNA molecule encodedby a gene. In some embodiments, the microorganisms can express exogenousgenes at levels high enough that permit harvesting products of theexogenous genes from the tumor. Expression of endogenous genes can becontrolled by a constitutive promoter, or by an inducible promoter.Expression can also be influenced by one or more proteins or RNAmolecules expressed by the microorganism. An exemplary induciblepromoter system can include a chimeric transcription factor containing aprogesterone receptor fused to the yeast GAL4 DNA-binding domain and tothe activation domain of the herpes simplex virus protein VP16, and asynthetic promoter containing a series of GAL4 recognition sequencesupstream of the adenovirus major late E1B TATA box, linked to one ormore exogenous genes; in this exemplary system, administration of RU486to a subject can result in induction of the exogenous genes. Exogenousgenes expressed can include genes encoding a therapeutic gene product,genes encoding a detectable gene product such as a gene product that canbe used for imaging, genes encoding a gene product to be harvested,genes encoding an antigen of an antibody to be harvested. Themicroorganisms provided herein can be used for expressing genes in vivoand in vitro. Exemplary proteins include reporter proteins (E. coliβ-galactosidase, β-glucuronidase, xanthineguaninephosphoribosyltransferase), proteins facilitating detection, i.e., adetectable protein or a protein capable of inducing a detectable signal,(e.g., luciferase, green and red fluorescent proteins, transferrinreceptor), proteins useful for tumor therapy (pseudomonas A endotoxin,diphtheria toxin, p53, Arf, Bax, tumor necrosis factor-alpha, HSV TK, E.coli purine nucleoside phosphorylase, angiostatin, endostatin, differentcytokines) and many other proteins.

iii. Detectable Gene Product

The microorganisms provided herein can express one or more genes whoseproducts are detectable or whose products can provide a detectablesignal. A variety of detectable gene products, such as detectableproteins are known in the art, and can be used with the microorganismsprovided herein. Detectable proteins include receptors or other proteinsthat can specifically bind a detectable compound, proteins that can emita detectable signal such as a fluorescence signal, enzymes that cancatalyze a detectable reaction or catalyze formation of a detectableproduct.

In some embodiments, the microorganism expresses a gene encoding aprotein that can emit a detectable signal or that can catalyze adetectable reaction. A variety of DNA sequences encoding proteins thatcan emit a detectable signal or that can catalyze a detectable reaction,such as luminescent or fluorescent proteins, are known and can be usedin the microorganisms and methods provided herein. Exemplary genesencoding light-emitting proteins include genes from bacterial luciferasefrom Vibrio harveyi (Belas et al., Science 218 (1982), 791-793),bacterial luciferase from Vibrio fischeri (Foran and Brown, Nucleicacids Res. 16 (1988), 177), firefly luciferase (de Wet et al., Mol.Cell. Biol. 7 (1987), 725-737), aequorin from Aequorea victoria (Prasheret al., Biochem. 26 (1987), 1326-1332), Renilla luciferase from Renillareniformis (Lorenz et al., PNAS USA 88 (1991), 4438-4442) and greenfluorescent protein from Aequorea victoria (Prasher et al., Gene 111(1987), 229-233). Transformation and expression of these genes inmicroorganisms can permit detection of microorganismal colonies, forexample, using a low light imaging camera. Fusion of the lux A and lux Bgenes can result in a fully functional luciferase protein (Escher etal., PNAS 86 (1989), 6528-6532). This fusion gene (Fab2) has introducedinto a variety of microorganisms followed by microorganismal infectionand imaging based on luciferase expression. In some embodiments,luciferases expressed in bacteria can require exogenously addedsubstrates such as decanal or coelenterazine for light emission. Inother embodiments, microorganisms can express a complete lux operon,which can include proteins that can provide luciferase substrates suchas decanal. For example, bacteria containing the complete lux operonsequence, when injected intraperitoneally, intramuscularly, orintravenously, allowed the visualization and localization of bacteria inlive mice indicating that the luciferase light emission can penetratethe tissues and can be detected externally (Contag et al., Mol.Microbiol. 18 (1995), 593-603).

In other embodiments, the microorganism can express a gene that can binda detectable compound or that can form a product that can bind adetectable compound. A variety of gene products, such as proteins, thatcan specifically bind a detectable compound are known in the art,including receptors, metal binding proteins, ligand binding proteins,and antibodies. Any of a variety of detectable compounds can be used,and can be imaged by any of a variety of known imaging methods.Exemplary compounds include receptor ligands and antigens forantibodies. The ligand can be labeled according to the imaging method tobe used. Exemplary imaging methods include any of a variety magneticresonance methods such as magnetic resonance imaging (MRI) and magneticresonance spectroscopy (MRS), and also include any of a variety oftomographic methods including computed tomography (CT), computed axialtomography (CAT), electron beam computed tomography (EBCT), highresolution computed tomography (HRCT), hypocycloidal tomography,positron emission tomography (PET), single-photon emission computedtomography (SPECT), spiral computed tomography and ultrasonictomography.

Labels appropriate for magnetic resonance imaging are known in the art,and include, for example, gadolinium chelates and iron oxides. Use ofchelates in contrast agents is known in the art. Labels appropriate fortomographic imaging methods are known in the art, and include, forexample, β-emitters such as ¹¹C, ¹³N, ¹⁵O or ⁶⁴Cu or (b) γ-emitters suchas ¹²³I. Other exemplary radionuclides that can be used, for example, astracers for PET include ⁵⁵Co, ⁶⁷Ga, ⁶⁸Ga, ⁶⁰Cu(II), ⁶⁷Cu(II), ⁵⁷Ni, ⁵²Feand ¹⁸F. Examples of useful radionuclide-labeled agents are ⁶⁴Cu-labeledengineered antibody fragment (Wu et al., PNAS USA 97 (2002), 8495-8500),⁶⁴Cu-labeled somatostatin (Lewis et al., J. Med. Chem. 42 (1999),1341-1347),⁶⁴Cu-pyruvaldehyde-bis(N4methylthiosemicarbazone)-(⁶⁴Cu-PTSM) (Adonai etal., PNAS USA 99 (2002), 3030-3035), ⁵²Fe-citrate (Leenders et al., J.Neural. Transm. Suppl. 43 (1994), 123-132), ⁵²Fe/^(52m)Mn-citrate(Calonder et al., J. Neurochem. 73 (1999), 2047-2055) and ⁵²Fe-labelediron (III) hydroxide-sucrose complex (Beshara et al., Br. J. Haematol.104 (1999), 288-295, 296-302).

iv. Therapeutic Gene Product

The microorganisms provided herein can express one or more genes whoseproducts cause cell death or whose products cause an anti-tumor immuneresponse, such genes can be considered therapeutic genes. A variety oftherapeutic gene products, such as toxic or apoptotic proteins, orsiRNA, are known in the art, and can be used with the microorganismsprovided herein. The therapeutic genes can act by directly killing thehost cell, for example, as a channel-forming or other lytic protein, orby triggering apoptosis, or by inhibiting essential cellular processes,or by triggering an immune response against the cell, or by interactingwith a compound that has a similar effect, for example, by converting aless active compound to a cytotoxic compound.

In some embodiments, the microorganism can express a therapeuticprotein. A large number of therapeutic proteins that can be expressedfor tumor treatment are known in the art, including, but not limited totumor suppressors, toxins, cytostatic proteins, and cytokines. Anexemplary, non-limiting list of such proteins includes WT1, p53, p16,Rb, BRCA1, cystic fibrosis transmembrane regulator (CFTR), Factor VIII,low density lipoprotein receptor, beta-galactosidase,alpha-galactosidase, beta-glucocerebrosidase, insulin, parathyroidhormone, alpha-1-antitrypsin, rsCD40L, Fas-ligand, TRAIL, TNF,antibodies, microcin E492, diphtheria toxin, Pseudomonas exotoxin,Escherichia coli Shig toxin, Escherichia coli Verotoxin 1, andhyperforin.

In other embodiments, the microorganism can express a protein thatconverts a less active compound into a compound that causes tumor celldeath. Exemplary methods of conversion of such a prodrug compoundinclude enzymatic conversion and photolytic conversion. A large varietyof protein/compound pairs are known in the art, and include, but are notlimited to Herpes simplex virus thymidine kinase/gancyclovir, varicellazoster thymidine kinase/gancyclovir, cytosine deaminase/5-fluorouracil,purine nucleoside phosphorylase/6-methylpurine deoxyriboside, betalactamase/cephalosporin-doxorubicin, carboxypeptidaseG2/4-[(2-chloroethyl)(2-mesuloxyethyl)amino]benzoyl-L-glutamic acid,cytochrome P450/acetominophen, horseradish peroxidase/indole-3-aceticacid, nitroreductase/CB1954, rabbitcarboxylesterase/7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin,mushroomtyrosinase/bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28,beta galactosidase/1-chloromethyl-5-hydroxy-1,2-dihyro-3H-benz[e]indole,beta glucuronidase/epirubicin glucuronide, thymidinephosphorylase/5′-deoxy-5-fluorouridine, deoxycytidine kinase/cytosinearabinoside, and linamerase/linamarin.

In another embodiment, the therapeutic gene product can be an siRNAmolecule. The siRNA molecule can be directed against expression of atumor-promoting gene, such as, but not limited to, an oncogene, growthfactor, angiogenesis promoting gene, or a receptor. The siRNA moleculealso can be directed against expression of any gene essential for cellgrowth, cell replication or cell survival. The siRNA molecule also canbe directed against expression of any gene that stabilizes the cellmembrane or otherwise limits the number of tumor cell antigens releasedfrom the tumor cell. Design of an siRNA can be readily determinedaccording to the selected target of the siRNA; methods of siRNA designand downregulation of genes are known in the art, as exemplified in U.S.Pat. Pub. No. 20030198627.

In one embodiment, the therapeutic compound can be controlled by aregulatory sequence. Suitable regulatory sequences which, for example,are functional in a mammalian host cell are well known in the art. Inone example, the regulatory sequence can contain a natural or syntheticvaccinia virus promoter. In another embodiment, the regulatory sequencecontains a poxvirus promoter. When viral microorganisms are used, stronglate promoters can be used to achieve high levels of expression of theforeign genes. Early and intermediate-stage promoters, however, can alsobe used. In one embodiment, the promoters contain early and latepromoter elements, for example, the vaccinia virus early/late promoterp7.5, vaccinia late promoter p11, a synthetic early/late vaccinia pE/Lpromoter (Patel et al., (1988), Proc. Natl. Acad. Sci. USA 85,9431-9435; Davison and Moss, (1989), J Mol Biol 210, 749-769; Davison etal., (1990), Nucleic Acids Res. 18, 4285-4286; Chakrabarti et al.,(1997), BioTechniques 23, 1094-1097).

v. Expressing a Superantigen

The microorganisms provided herein can be modified to express one ormore superantigens. Superantigens are antigens that can activate a largeimmune response, often brought about by a large response of T cells. Avariety of superantigens are known in the art including, but not limitedto, diphtheria toxin, staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2,SED, SEE and SEH), Toxic Shock Syndrome Toxin 1, Exfoliating Toxins(EXft), Streptococcal Pyrogenic Exotoxin A, B and C (SPE A, B and C),Mouse Mammary Tumor Virus proteins (MMTV), Streptococcal M proteins,Clostridial Perfringens Enterotoxin (CPET), mycoplasma arthritissuperantigens.

Since many superantigens also are toxins, if expression of amicroorganism of reduced toxicity is desired, the superantigen can bemodified to retain at least some of its superantigenicity while reducingits toxicity, resulting in a compound such as a toxoid. A variety ofrecombinant superantigens and toxoids of superantigens are known in theart, and can readily be expressed in the microorganisms provided herein.Exemplary toxoids include toxoids of diphtheria toxin, as exemplified inU.S. Pat. No. 6,455,673 and toxoids of Staphylococcal enterotoxins, asexemplified in U.S. Pat. Pub. No. 20030009015.

vi. Expressing a Gene Product to be Harvested

Exemplary genes expressible by a microorganism for the purpose ofharvesting include human genes. An exemplary list of genes includes thelist of human genes and genetic disorders authored and edited by Dr.Victor A. McKusick and his colleagues at Johns Hopkins University andelsewhere, and developed for the World Wide Web by NCBI, the NationalCenter for Biotechnology Information. Online Mendelian Inheritance inMan, OMIM™. Center for Medical Genetics, Johns Hopkins University(Baltimore, Md.) and National Center for Biotechnology Information,National Library of Medicine (Bethesda, Md.), 1999, and those availablein public databases, such as PubMed and GenBank (see, e.g.,(ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM) These genes include, butare not limited to: 239f2h9, 3pk, 4ebp1, 4ebp2, a11, a12m1, a12m2,a12m3, a12m4, a15, a1b, a1bg, a1st, a2m, a2mr, a2mrap, aa, aaa, aaa,aabt, aac1, aac2, aact, aadac, aanat, aars, aas, aat, aavs1, abc1, abc2,abc3, abc7, abc8, abcr, abi1, abl1, abl2, abl1, abo, abp, abp1, abpa,abpx, abr, acaa, acac, acaca, acacb, acad1, acadm, acads, acadsb,acadv1, acat, acat1, acat2, acc, accb, accn1, accn2, accpn, ace1, ach,ache, achm1, achm2, achrb, achrd, achrg, acls, acly, aco1, aco2, acox,acox1, acox2, acox3, acp1, acp2, acp5, acpp, acr, acrv1, acs3, acs3,acs4, act2, act35, acta1, acta2, acta3, actb, actc, actg1, actg2, act11,actn1, actn2, actn3, actsa, acug, acvr1, acvr2b, acvr11, acvrik1,acvrlk2, acvrlk3, acy1, ad1, ad2, ad3, ad4, ad5, ada, adam10, adam11,adam12, adam3, adam3a, adam3b, adam8, adar, adarb1, adarb2, adcp1,adcp2, adcy1, adcy2, adcy3, adcy3, adcy4, adcy5, adcy6, adcy7, adcy8,adcy9, adcyap1, adcyaplr1, add1, add2, add3, add1, adfn, adh1, adh2,adh3, adh4, adh5, adh7, adhaps, adhc1, adhr, adhr, adk, ad1, adm, adm1x,adora1, adora2a, adora2b, adora21, adora21, adora3, adprt, adra1a,adra1b, adra1c, adra1d, adra2a, adra2b, adra2c, adra211, adra212,adra2r, adrb1, adrb1r, adrb2, adrb2r11, adrb3, adrbk1, adrbk2, ads1,adss, adtb1, adx, adxr, ae1, ae2, ae3, aeg11, aemk, aes, af10, af17,af4, af6, af8t, af9, afd1, afdn, afg3, afg311, afm, afp, afx1, aga,agc1, ager, ag1, agmx1, agmx2, agp1, agp7, agps, agrn, agrp, agrt, ags,agt, agti1, agtr1, agtr1a, agtr2, agtr11, agxt, ahc, ahcy, ahd, ahds,ahnak, aho2, ahr, ahsg, ahx, aib1, aic, aic1, aied, aih1, aih2, aih3,aim1, air, airc, aire, ak1, ak2, ak3, akap149, akt1, akt2, aku, alad,alas1, alas2, alb, alb2, alba, alcam, ald, aldh1, aldh10, aldh2, aldh3,aldh4, aldh5, aldh6, aldh9, aid 11, aldoa, aldob, aldoc, aldr1, alds,alk, alk1, alk2, alk3, alk6, alms1, alox12, alox15, alox5, alp, alpi,alp1, alpp, alpp12, alr, alr, als1, als2, als4, als5, alss, ambn, ambp,amcd1, amcd2b, amcn, amcn1, amcx1, amd1, amdm, amelx, amely, amfr, amg,amg1, amgx, amh, amhr, amhr2, am11, am11t1, am12, am13, amog, ampd1,ampd2, ampd3, amph, amph1, ampk, amt, amy1a, amy1b, amy1c, amy2a, amy2b,an2, anc, ancr, ang, ang1, anh1, ank1, ank2, ank3, anop1, anova, anp,anpep, anpra, anprb, anprc, ans, ant1, ant2, ant3, ant3y, anx1, anx11,anx13, anx2, anx214, anx3, anx4, anx5, anx6, anx7, anx8, aoah, aoc2,aox1, ap2tf, apah1, apba1, apba2, apbb1, apbb2, apc, apcs, ape, apeced,apeh, apex, api1, api2, api3, apj, aplp, aplp1, aplp2, apnh, apo31,apoa1, apoa2, apoa4, apob, apobec1, apoc1, apoc2, apoc3, apoc4, apod,apoe, apoer2, apoh, apolmt, apolp1, apolp2, app, appbp1, app11, aprf,aprt, aps, apt1, apt11g1, apx1, apy, aqdq, aqp0, aqp1, aqp2, aqp21,aqp3, aqp4, aqp5, aqp6, aqp7, ar, ar1, ara, araf1, araf2, arcn1, ard1,ard1, areg, arf1, arf2, arf3, arf41, arf5, arg, arg1, args, arh12, arh6,arh9, arha, arhb, arhc, arhg, arhgap2, arhgap3, arhgap6, arhgdia,arhgdib, arhh, arix, ar12, armd1, arnt, arnt1, aro, arp, arp1, arpkd,arr3, arrb1, arrb2, arsa, arsacs, arsb, arsc1, arsc2, arsd, arse, arsf,art, art1, art3, art4, arts, arvd1, arvd2, arvd3, arvd4, as, asat, asb,arcc11, asc12, asct1, asd1, asd2, asgr1, asgr2, ash1, asip, as1, as1n,asm1, asma, asmd, asmt, asmt1x, asmty, asnrs, asns, aspa, ass, astm1,astn, asv, at, at1, at2r1, at3, ata, atbf1, atcay, atf1, ath1, aths,atm, atoh1, atox1, atp1a1, atp1a2, atp1a3, atp1a11, atp1b1, atp1b2,atp1b3, atp1b11, atp1g1, atp2a1, atp2a2, atp2a3, atp2b, atp2b1, atp2b2,atp2b2, atp2b3, atp2b4, atp4a, atp4b, atp5, atp5a, atp5b, atp5g1,atp5g2, atp5g3, atp5o, atp6a, atp6b1, atp6c, atp6e, atp6n1, atp7a,atp7b, atpm, atpsb, atpsk1, atpsk2, atq1, atr, atr, atr1, atr1, atr2,atrc1, atrc2, atrx, ats, atsv, atx1, atx2, au, auf1, auf1a, aut, avcd,aved, avp, avprl1a, avpr1b, avpr2, avpr3, avrp, avsd, awa1, ax1, ax11g,axsf, azf1, azf2, azgp1, azu1, b120, b144, b1g1, b29, b2m, b2mr,b3galt4, b4galt1, ba2r, bab1, bag1, bai1, bai2, bai3, bak1, bam22, bap1,bap135, bapx1, bard1, bark2, bas, bat1, bat2, bat3, bat4, bat5, bax,bb1, bbbg1, bbbg2, bbs1, bbs2, bbs3, bbs4, bbs5, bcas1, bcat1, bcat2,bcate2, bcd1, bcei, bche, bckdha, bckdhb, bc11, bc110, bc12, bc12a1,bc1212, bc13, bc15, bc16, bc17, bc17a, bc18, bc19, bc81w, bcm, bcm1,bcma, bcns, bcns, bcp, bcpm, bcpr, bcr, bcr12, bcr13, bcr14, bcsg1,bct1, bct2, bdb, bdb1, bdc, bde, bdkrb1, bdkrb2, bdmf, bdmr, bdnf, bed,bedp, bek, bene, bevi, bf, bf1, bf2, bfhd, bfic, bfls, bfnc2, bfp,bfsp1, bft, bglap, bgmr, bgn, bgp, bhd, bhpcdh, bhr1, bicd1, bid, bigh3,bin1, bir, bjs, bkma1, blast1, blau, blk, blm, blmh, bltr, blvra, blvrb,blym, bmal1, bmd, bmh, bmi1, bmp1, bmp2, bmp2a, bmp2b1, bmp3, bmp4,bmp5, bmp6, bmp7, bmp8, bmpr1a, bmpr1b, bmx, bmyb, bn51t, bnc, bnc1,bnp, bor, bpad, bpag1, bpag2, bpes, bpes1, bpes2, bpgm, bph1, bpi, br,br140, braf, brca1, brca2, brca3, brcacox, brcd1, brcd2, brdt, brf1,brhc, bric, brks, brn3a, brn3b, brn3c, brrn1, brw1c, bs, bsap, bsep,bsf2, bsg, bsnd, bss1, bst1, bst2, btak, btc, btd, bteb, bteb1, btg1,btg2, bths, btk, btk1, btn, bts, bub1b, bubr1, bwr1a, bwr1b, bws,bwscr1a, bwscr1b, bzrp, bzx, c11orf13, c1nh, c1qa, c1qb, c1qbp, c1qg,c1r, c1s, c2, c21orf1, c21orf2, c21orf3, c2ta, c3, c3br, c3dr, c3g, c4a,c4b, c4bpa, c4bpb, c4f, c4s, c5, c5ar, c5r1, c6, c7, c8a, c8b, c8g, c9,ca1, ca12, ca125, ca2, ca21h, ca3, ca4, ca5, ca6, ca7, ca8, ca9, caaf1,cabp9k, cac, cac, caca, cacd, cacna1a, cacna1b, cacna1c, cacna1d,cacna1e, cacna1f, cacna1s, cacna2, cacnb1, cacnb2, cacnb3, cacnb4,cacng, cacnl1a1, cacnl1a2, cacnl1a3, cacnl1a4, cacnl1a5, cacnl1a6,cacnl2a, cacnlb1, cacnlg, cacp, cact, cacy, cad, cad11, cadasi1, cae1,cae3, caf, caf1a, caga, cagb, cain, cak, cak1, cal11, calb1, calb2,calb3, calc1, calc2, calca, calcb, calcr, calb1, calla, calm1, calm2,calm3, calm11, calm13, calna, calna3, calnb, calnb1, calr, cals, calt,calu, cam, camk4, camkg, caml1, camlg, camp, can, canp3, canx, cap2,cap3, cap37, capb, capg, cap1, capn1, capn2, capn3, capn4, cappa2,cappb, capr, caps, capza2, capzb, car, carp, cars, cart1, cas, cas2,casi1, casp1, casp10, casp2, casp3, casp3, casp4, casp5, casp6, casp7,casp8, casq1, casq2, casr cast, cat, cat1, cat4, catf1, catm, cav1,cav2, cav3, cbbm, cbd, cbfa1, cbfa2, cbfa2t1, cbfa3, cbfb, cbg, cbl,cbl2, cbln2, cbp, cbp, cbp2, cbp68, cbr1, cbs, cbt, cbt1, cc10, cca,cca1, cca11, cca12, ccb11, ccckr5, ccg1, ccg2, cch11a1, cch11a2,cch11a3, cch1b1, cck, cckar, cckbr, cc1, ccm1, ccm2, ccm3, ccn1, ccna,ccnb1, ccnc, ccnd1, ccnd2, ccnd3, ccne, ccnf, ccng1, ccnh, ccnt, ccnt1,cco, ccr10, ccr2, ccr3, ccr9, ccsp, cct, ccv, cczs, cd, cd10, cd11a,cd11b, cd11c, cd13, cd137, cd14, cd15, cd151, cd156, cd16, cd164, cd18,cd19, cd1a, cd1b, cd1c, cd1d, cd1e, cd2, cd20, cd22, cd23, cd24, cd26,cd27, cd271, cd28, cd281g, cd281g2, cd30, cd32, cd33, cd34, cd36,cd3611, cd3612, cd37, cd38, cd39, cd3911, cd3d, cd3e, cd3g, cd3z, cd4,cd40, cd401g, cd41b, cd43, cd44, cd45, cd46, cd47, cd48, cd49b, cd49d,cd5, cd53, cd57, cd58, cd59, cd51, cd6, cd63, cd64, cd68, cd69, cd7,cd70, cd71, cd72, cd74, cd79a, cd79b, cd80, cd81, cd82, cd82, cd86,cd8a, cd8b, cd8b1, cd9, cd94, cd95, cd97, cd99, cda, cda1, cda3, cdan1,cdan2, cdan3, cdb2, cdc2, cdc20, cdc25a, cdc25b, cdc25c, cdc27, cdc211,cdc212, cdc214, cdc34, cdc42, cdc51, cdc7, cdc711, cdcd1, cdcd2, cdcd3,cdc11, cdcre1, cdg1, cdgd1, cdgg1, cdgs2, cdh1, cdh11, cdh12, cdh13,cdh14, cdh15, cdh16, cdh16, cdh17, cd2, cdh3, cdh3, cdh5, cdh7, cdh8,cdhb, cdhh, cdhp, cdhs, cdk2, cdk3, cdk4, cdk5, cdk7, cdk8, cdk9, cdkn1,cdkn1a, cdkn1b, cdkn1c, cdkn2a, cdkn2b, cdkn2d, cdkn3, cdkn4, cd11, cdm,cdmp1, cdmt, cdpx1, cdpx2, cdpxr, cdr1, cdr2, cdr3, cdr62a, cdsn, cdsp,cdtb, cdw50, cdx1, cdx2, cdx3, cdx4, cea, cebp, cebpa, cebpb, cebpd,cebpe, cecr, ce1, ce11, cen1, cenpa, cenpb, cenpc, cenpc1, cenpe, cenpf,cerd4, ces, ces1, cetn1, cetp, cf, cf2r, cfag, cfag, cfc, cfd1, cfeom1,cfeom2, cfh, cf11, cf12, cfnd, cfns, cftr, cg1, cga, cgat, cgb, cgd,cgf1, cgh, cgrp, cgs23, cgt, cgthba, chac, chat, chc1, chd1, chd2, chd3,chd4, chd5, chdr, che1, che2, ched, chek1, chga, chgb, chgc, chh,chi311, chip28, chit, chk1, chlr1, chlr2, chm, chm1, chn, chn1, chn2,chop10, chr, chr39a, chr39b, chr39c, chrm1, chrm2, chrm3, chrm4, chrm5,chrna1, chrna2, chrna3, chrna4, chrna5, chrna7, chrnb1, chrnb2, chrnb3,chrnb4, chrnd, chrne, chrng, chrs, chs1, chx10, ciipx, cip1, cirbp,cish, ck2a1, ckap1, ckb, ckbb, ckbe, ckm, ckmm, ckmt1, ckmt2, ckn1,ckn2, ckr3, ckr11, ckr13, cl, cl100, cla1, cla1, clac, clapb1, clapm1,claps3, clc, clc7, clck2, clcn1, clcn2, clcn3, clcn4, clcn5, clcn6,clcn7, clcnka, clcnkb, cld, cldn3, cldn5, clg, clg1, clg3, clg4a, clg4b,cli, clim1, clim2, clk2, clk3, cln1, cln2, cln3, cln5, cln6, cln80,clns1a, clns1b, clp, clpp, clps, clta, cltb, cltc, cltc11, cltd, clth,clu, cma1, cmah, cmar, cmd1, cmd1a, cmd1b, cmd1c, cmd1d, cmd1e, cmd1f,cmd3a, cmdj, cmh1, cmh2, cmh3, cmh4, cmh6, cmkbr1, cmkbr2, cmkbr3,cmkbr5, cmkbr6, cmkbr7, cmkbr8, cmkbr9, cmkbr12, cmklr1, cmkr11, cmkr12,cml, cmm, cmm2, cmoat, cmp, cmpd1, cmpd2, cmpd2, cmpd3, cmpx1, cmt1a,cmt1b, cmt2a, cmt2b, cmt2d, cmt2d, cmt4a, cmt4b, cmtnd, cmtx1, cmtx2,cna1, cna2, cnbp1, cnc, cncg1, cncg2, cncg31, cnd, cng3, cnga1, cnga3,cngb1, cnn1, cnn2, cnn3, cnp, cnr1, cnsn, cntf, cntfr, cntn1, co, coca1,coca2, coch, cod1, cod2, coh1, coil, col10a1, col11a1, col11a2,col12a11, col13a1, col15a1, col16a1, col17a1, col18a1, col19a1, col1a1,col1a2, col1ar, col2a1, col3a1, col4a1, col4a2, col4a3, col4a4, col4a5,col4a6, col5a1, col5a2, col6a1, col6a2, col6a3, col7a1, col8a1, col8a2,col9a1, col9a1, col9a2, col9a3, colq, comp, comt, copeb, copt1, copt2,cord1, cord2, cord5, cord6, cort, cot, cox10, cox4, cox5b, cox6a1,cox6b, cox7a1, cox7a2, cox7a3, cox7am, cox8, cp, cp107, cp 115, cp20,cp47, cp49, cpa1, cpa3, cpb2, cpb2, cpd, cpe, cpetr2, cpm, cpn, cpn1,cpn2, cpo, cpp, cpp32, cpp32, cppi, cps1, cpsb, cpsd, cpt1a, cpt1b,cpt2, cpu, cpx, cpx, cpxd, cr1, cr2, cr3a, crabp1, crabp2, crapb, crarf,crat, crbp1, crbp2, crd, crd1, creb1, creb2, crebbp, creb11, crem,crfb4, crfr2, crh, crhbp, crhr, crhr1, crhr2, crip, crk, crk1, crm1,crmp1, crmp2, crp, crp1, crs, crs1c, crs2, crs3, crsa, crt, crtl1, crtm,crx, cry1, cry2, crya1, crya2, cryaa, cryab, cryb1, cryb2, cryb3,cryba1, cryba2, cryba4, crybb1, crybb2, crybb3, cryg1, cryg2, cryg3,cryg4, cryg8, cryg, cryga, crygb, crygc, crygd, crygs, crym, cryz, cs,csa, csb, csbp1, csci, csd, csd2, csda, cse, csel1, csf1, csf1r, csf2,csf2ra, csf2rb, csf2ry, csf3, csf3r, csh1, csh2, csk, csmf, csn1, csn10,csn2, csn3, csnb1, csnb2, csnb3, csnk1a1, csnk1d, csnk1e, csnk1g2,csnk2a1, csnk2a2, csnk2b, csnu3, cso, cspb, cspg1, cspg2, cspg3, csr,csrb, csrp, csrp1, csrp2, cst1, cst1, cst2, cst3, cst4, cst4, cst5,cst6, csta, cstb, csx, ct2, ctaa1, ctaa2, ctag, ctb, ctbp1, ctbp2, ctgf,cth, cthm, ctk, ctla1, ctla3, ctla4, ctla8, ctm, ctnna1, ctnna2, ctnnb1,ctnnd, ctnnd1, ctnr, ctns, ctp, ctpct, ctps, ctr1, ctr2, ctrb1, ctr1,ctsa, ctsb, ctsc, ctsd, ctse, ctsg, ctsg12, ctsh, ctsk, cts1, ctss,ctsw, ctsz, ctx, cubn, cul3, cul4b, cul5, cutl1, cvap, cvd1, cv1, cx26,cx31, cx32, cx37, cx40, cx43, cx46, cx50, cxb3s, cxcr4, cxorf4, cyb5,cyb561, cyba, cybb, cyc1, cyk4, cyld1, cymp, cyp1, cyp11a, cyp11b1,cyp11b2, cyp17, cyp19, cyp1a1, cyp1a2, cyp1b1, cyp21, cyp24, cyp27,cyp27a1, cyp27b1, cyp2a, cyp2a3, cyp2a6, cyp2b, cyp2c, cyp2c19, cyp2c9,cyp2d, cyp2d, cyp2e, cyp2e1, cyp2f1, cyp2j2, cyp3a4, cyp4a11, cyp4b1,cyp51, cyp7, cyp7a1, cyr61, cyrn1, cyrn2, czp3, d10s105e, d10s170,d10s170, d11s302e, d11s636, d11s813e, d11s833e, d12s2489e, d12s53e,d13s1056e, d13s25, d14s46e, d15s12, d15s226e, d15s227e, d16s2531e,d16s469e, d17s136e, d17s811e, d18s892e, d19s204, d19s381e, d1s111,d1s155e, d1s166e, d1s1733e, d1s2223e, d1s61, d2h, d2s201e, d2s448,d2s488e, d2s69e, d3s1231e, d3s1319e, d3s48e, d4, d4s90, d5s1708, d5s346,d6, d6s1101, d6s207e, d6s2245e, d6s228e, d6s229e, d6s230e, d6s231e,d6s51e, d6s52e, d6s54e, d6s81e, d6s82e, d7s437, d8s2298e, d9s46e, da1,da2b, dab2, dac, dad1, daf, dag, dag1, dag2, dagk1, dagk4, dam10, dam6,damox, dan, dao, dap, dap3, dap5, dapk1, dar, dat1, dax1, daxx, daz,dazh, daz1, dba, dbccr1, dbcn, dbh, dbi, dbi, dbl, dbm, dbn1, dbp, dbp,dbp1, dbp2, dbpa, dbt, dbx, dby, dcc, dce, dci, dck, dcn, dcoh, dcp1,dcr, dcr3, dct, dctn1, dcx, ddb1, ddb2, ddc, ddh1, ddh2, ddit1, ddit3,ddost, ddp, ddpac, ddr, ddx1, ddx10, ddx11, ddx12, ddx15, ddx16, ddx2a,ddx3, ddx5, ddx6, ddx9, dec, decr, def1, def4, def5, def6, defa1, defa4,defa5, defa6, defb1, defb2, dek, denn, dents, dep1, der12, des, dff1,dffa, dffrx, dffry, dfn1, dfn2, dfn3, dfn4, dfn6, dfna1, dfna10, dfna11,dfna12, dfna13, dfna2, dfna2, dfna4, dfna5, dfna6, dfna7, dfna8, dfna9,dfnb1, dfnb12, dfnb13, dfnb14, dfnb16, dfnb17, dfnb18, dfnb2, dfnb3,dfnb4, dfnb5, dfnb6, dfnb7, dfnb8, dfnb9, dgcr, dgcr2, dgcr2, dgcr6,dgi1, dgka, dgkq, dgpt, dgpt, dgs, dgs2, dgsi, dgu, dhc2, dhcr7, dhfr,dhlag, dhp, dhpr, dhps, dhrd, dhtr, di, di1, dia, dia1, dia2, dia4,diaph1, diaph2, dif2, diff6, dipi, dir, dkc, dkc1, dlc1, dld, dlg1,dlg2, dlg3, dlg4, dlst, dlx1, dlx2, dlx2, dl3, dlx4, dlx5, dlx6, dlx7,dlx8, dm, dm2, dmahp, dmbt1, dmd, dmda1, dmd1, dmh, dmk, dmp1, dmpk,dmsfh, dmt, dmt1, dmtn, dna21, dnah, dnah1, dnah11, dnah12, dnah2,dnahc1, dnahc11, dnahc2, dnahc3, dnase1, dnase111, dnase113, dnase2,dnch2, dnc1, dncm, dnec1, dne11, dn1, dn11, dn111, dnm1, dnmt1, dnmt2,dnpk1, dns, dntt, do, doc1, doc2, dock1, dock180, dod, dok1, dom, dp1,dp1, dp2, dp3, dpagt2, dpc4, dpd, dpde1, dpde2, dpde3, dpde4, dpep1,dph212, dpp, dpp4, dpp6, dpt, dpyd, dpys, dpys11, dpys12, dr1, dr3,dr31g, dr5, dra, drad, drada, dra1, drd1, drd1b, drd1b, drd112, drd2,drd3, drd4, drd5, dri11, drp1, drp1, drp2, drp2, drp3, drp1a, drt, dsc1,dsc2, dsc3, dsc3, dsc4, dscam, dscr, dsg1, dsg2, dsg3, dsp, dspg3, dspp,dss, dss1, dtd, dtdp2, dtdst, dtna, dtr, dts, dus, dusp1, dusp11, dusp2,dusp3, dusp4, dusp5, dusp6, dusp7, dusp8, dut, dv1, dv11, dv11, dv13,dxf68s1e, dxs1272e, dxs128, dxs1283e, dxs423e, dxs435e, dxs522e, dxs648,dxs707, dxs8237e, dxys155e, dylx2, dyrk, dys, dysf, dyt1, dyt3, dyt5,dyt6, dyt7, dyt8, dyt9, dyx1, dyx2, e11s, e14, e1b, e2a, e2f1, e2f2,e2f3, e2f4, e3, e4f, e4f1, e4tf1a, e4tf1b, ea1, eaac1, eaat1, eaat2,eac, ead, eag, eap, ear1, ear2, ear3, ebaf, ebf, ebi1, ebm, ebn1, ebn1,ebn2, ebr2a, ebs1, ebvm1, ebvs1, ec1, eca1, ecb2, ece1, ecgf1, ech1,echs1, eck, ecm1, ecp, ecs1, ect2, ed1, ed2, ed3, ed4, eda, eda3, eddr1,edg3, edg6, edh, edh17b2, edh17b2, edh17b3, edm1, edm2, edm3, edmd,edmd2, edn, edn1, edn2, edn3, ednra, ednrb, eec1, eec2, eef1a1, eef1a2,eef1b1, eef1b2, eef1b3, eef1b4, eef2, eeg1, eegv1, eek, een, ef1a, ef2,efe2, efemp1, efl6, efmr, efna1, efna3, efna4, efnb1, efnb2, efnb3, efp,eftu, egf, egfr, egi, egr1, egr2, egr3, egr4, ehhadh, ehoc1, ei, eif1a,eif2g A, eif2s3 A, eif3s10, eif3s6, eif4a1, eif4a2, eif4c, eif4e,eif4ebp1, eif4e2, eif4e11, eif4e12, eif4g, eif4g1, eif4g2, eif5a, ejm1,el1, ela1, ela2, elam1, elanh2, elav11, elav12, elav14, elc, ele1, elf3,elk1, elk2, elk3, elk4, ell, eln, em9, emap, emap1, emd, emd2, emk 1,emp1, emp55, emr1, ems1, emt, emtb, emx1, emx2, en1, en2, ena78, end,endog, enfl2, eng, en1, eno1, eno2, eno3, enpep, ent1, entk, enur1,enur2, enx2, eos, ep3, ep300, epa, epb3, epb311, epb41, epb4112, epb42,epb49, epb72, epha1, epha2, epha3, epha8, ephb1, ephb2, ephb3, ephb4,ephb6, epht1, epht2, epht3, ephx1, ephx2, epim, eplg1, eplg2, eplg3,eplg4, eplg5, eplg8, epm1, epm2, epm2a, epmr, epo, epor, eppk, eprs,eps15, eps8, ept, erba1, erba2, erba12, erba13, erbb2, erbb3, erbb4,erc55, ercc1, ercc2, ercc3, ercc4, ercc5, ercc6, ercm1, erda1, erf1,erg, erg3, ergic53, erh, erk, erk1, erk2, erk3, erm, erp11, erv1, erv1,erv3, ervr, ervt1ervt2, ervt3, ervt4, ervt5, eryf1, es1, es130, esa,esa1, esa4, esat, esb3, esd, esg, esr, esr1, esr2, esr11, esr12, esrra,esrrb, esrrg, ess1, est, est, est2, est25263, esx, etfa, etfb, etfdh,etk1, etk2, etm1, etm2, eto, ets1, ets2, etv1, etv3, etv4, etv5, etv6,evc, evc1, evda, evdb, evi1, evi2, evi2a, evi2b, evp1, evr1, evx1, evx2,ews, ewsr1, exlm1, ext1, ext2, ext3, ext11, ext12, eya1, eya2, eya3,eyc11, eyc13, ezh1, ezh1, ezh2, f10, f11, f12, f13a, f13a1, f13b, f2,f2r, f2r12, f2r13, f3, f5, f5f8d, f7, f7e, f7r, f8a, f8b, f8c, f8vwf,f9, fa, fa1, faa, fabp1, fabp2fabp3, fabp4, fabp6, fac1, faca, facc,facd, face, fac11, fac12, fac13, fac14, facv11, fad, fadd, fadk, fah,fak2, faldh, fal139, falz, fanca, fancc, fancd, fance, fancg, fap, fapa,farr, fas, fas1, fasn, fast1, fat, fau, fbln1, fbln2, fbn1, fbn2, fbn1,fbp1, fcar, fcc1, fce, fce2, fcer1a, fcer1b, fcer1g, fcer2, fcgr1a,fcgr1b, fcgr1c, fcgr2a, fcgr3a, fcgrt, fcmd, fcn1, fcn2, fcp, fcp1,fcpx, fct3a, fdc, fdft1, fdh, fdps11, fdps12, fdps13, fdps14, fdps15,fdx1, fdxr, fe65, fe6511, fea, feb1, feb2, fecb, fech, fen1, feo, feom,feom1, feom2, fer, fes, fet1, fevr, ffm, fga, fgarat, fgb, fgc, fgd1,fgdy, fgf1, fgf10, fgf11, fgf12, fgf13, fgf14, fgf2, fgf2, fgf3, fgf4,fgf5, fgf6, fgf7, fgf8, faf9, fgfa, fgfb, fgfr1, fgfr2, fgfr3, fgfr4,fgg, fgr, fgs1, fh, fh, fh3, fhc, fnf1, fhf3, fhf4, fhh2, fhit, fh11,fh12, fhr2, fic1, figf, fih, fim, fim1, fim3, fimg, fkbp12, fkbp1a,fkbp2, fkh2, fkh11, fkh110, fkh112, fkh115, fkh116, fkh117, fkh12,fkh15, fkh16, fkh17, fkh18, fkh19, fkhr, fkhr11, flg, flil, flii, fln1,fln2, flna, flnb, flnms, flot2, flt1, flt2, flt3, flt4, fmf, fmn, fmo1,fmo2, fmo3, fmod, fmr1, fmr2, fms, fl1, fn12, fnra, fnrb, fnrb1, fnta,fntb, folh, folh1, folr1, folr2, folt, fos, fosb, fos11, fos12, fpah,fpc, fpd1, fpdmm, fpf, fpgs, fp1, fpp, fpr1, fprh1, fprh2, fpr11, fpr12,fprp, fps12, fps13, fps14, fps15, fr, frap1, fraxa, fraxe, fraxf, frda,freac2, freac6, freac9, frg1, frp1, frv1, frv2, frv3, fsg1, fsgs, fshb,fshd1a, fshmd1a, fshprh1, fshr, fssv, fth1, fth16, ft1, ftz1, ftzf1,fuca1, fuca2, fur, fus, fuse, fut1, fut2, fut3, fut4, fut5, fut6, fut7,fut8, fvt1, fxr1, fxy, fy, fyn, fzd1, fzd2, fzd3, fzd5, fzd6, fzd7, fzr,g0s8, g10p1, g10p2, g17, g17p1, g19p1, g1p1, g1p2, g1p3, g22p1, g6pc,g6pd, g6pd1, g6pd1, g6pt, g6pt1, g6s, g7p1, ga2, gaa, gabatr, gabpa,gabpb1, gabra1, gabra2, gabra3, gabra4, gabra5, gabra6, gabrb1, gabrb2,gabrb3, gabrd, gabre, gabrg1, gabrg2, gabrg3, gabrr1, gabrr2, gad1,gad2, gad3, gadd153, gadd45, gak, gal, galbp, galc, gale, galgt, galk1,galk2, galn, galnact, galnr, galnr1, galns, galnt1, galnt2, galnt3,galr1, galt, gan, gan1, ganab, ganc, gap, gap1m, gap43, gapd, gar22,garp, gars, gart, gas, gas1, gas2, gas41, gas6, gas7, gasr, gast, gata1,gata2, gata3, gata4, gata6, gay1, gba, gbas, gbbb1, gbbb2, gbe1, gbp1,gbx2, gc, gcap, gcap2, gcdh, gcf1, gcf2, gcfx, gcg, gcgr, gch1, gck,gckr, gcn511, gcn512, gcnf, gcnt1, gcnt2, gcp, gcp2, gcs, gcs1, gcsf,gcsfr, gcsp, gctg, gcy, gda, gde, gdf5, gdf8, gdh, gdi1, gdi2, gdid4,gdld, gdnf, gdnfr, gdnfra, gdnfrb, gdx, gdxy, ge, gem, geney, gey, gf1,gf1, gfap, gfer, gfer, gfi1, gfpt, gfra1, gfra2, ggcx, ggt1, ggt2,ggta1, ggtb1, ggtb2, gh1, gh2, ghc®, ghdx, ghn, ghr, ghrf, ghrh, ghrhr,ghs, ghv, gif, gifb, gip, gip, gipr, girk1, girk2, girk3, girk4, gja1,gja3, gja4, gja5, gja8, gjb1, gjb2, gjb3, gk, gk2, gla, glat, gib1,glb2, glc1a, glc1b, glc1c, glc1d, glc1f, glc3a, glc3b, gic1c, gic1r,glct2, glct3, gldc, glepp1, glg1, gli, gli2, gli3, gli4, glnn, glns,glo1, glo2, glp1r, glra1, glra2, glra3, glrb, glrx, gls, glud1, glud2,glu1, glur1, glur2, glur3, glur4, glur5, glur6, glur7, glut1, glut2,glut3, glut4, glut5, glvr1, glvr2, gly96, glya, glyb, glys1, glyt1,glyt1, glyt2, gm2a, gma, gmcsf, gmds, gm1, gmpr, gmps, gnal1, gnal5,gnal6, gnai1, gnai2, gnai2a, gnai2b, gnai21, gnai3, gna1, gnao1, gnaq,gnas, gnas1, gnat1, gnat2, gnaz, gnb1, gnb2, gnb3, gng5, gn11, gnpta,gnrh1, gnrh2, gnrhr, gns, gnt1, golga4, got1, got2, gp130, gp1ba, gp1bb,gp2, gp2b, gp39, gp3a, gp75, gp78, gp9, gpa, gpam, gpat, gpb, gpc, gpc1,gpc3, gpc4, gpd, gpd1, gpd2, gpds1, gpe, gpi, gpi2, gpm6a, gpm6b, gpoa,gpr1, gpr10, gpr11, gpr12, gpr13, gpr15, gpr17, gpr18, gpr19, gpr2,gpr20, gpr21, gpr22, gpr23, gpr25, gpr29, gpr3, gpr30, gpr31, gpr32,gpr35, gpr37, gpr39, gpr4, gpr5, gpr6, gpr7, gpr8, gpr9, gprcy4, gprk21,gprk4, gprk5, gprk6, gprv28, gpsa, gpsc, gpt, gpx1, gpx2, gpx3, gpx4,gr2, grb1, grb10, grb2, grf2, gria1, gria2, gria3, gria4, grid2, grik1,grik2, grik3, grik4, grik5, grin1, grin2a, grin2b, grin2c, grin2d,grina, grk1, grk5, grk6, gr1, gr111, grm3, grm8, grmp, grn, gro1, gro2,gro3, grp, grp58, grp78, grpr, grx, gs, gs1, gsas, gsc, gsc1, gse, gshs,gs1, gsm1, gsn, gsp, gspt1, gsr, gss, gst12, gst11, gst2, gst2, gst3,gst4, gst5, gsta1, gsta2, gstm1, gstm11, gstm2, gstm3, gstm4, gstm5,gstp1, gstt2, gt1, gt335, gta, gtb, gtbp, gtd, gtf2e2, gtf2f1, gtf2h1,gtf2h2, gtf2h4, gtf2i, gtf2s, gtf3a, gtg, guc1a2, guc1a3, guc1b3, guc2c,guc2d, guc2f, guca1a, guca1b, guca2, guca2, guca2a, guca2b, gucsa3,gucsb3, gucy1a2, gucy1a3, gucy1b3, gucy2c, gucy2d, gucy2f, guk1, guk2,gulo, gulop, gusb, gusm, gust, gxp1, gypa, gypb, gypc, gype, gys, gys1,gys2, gzma, gzmb, gzmh, gzmm, h, h142t, h19, h1f0, h1f1, h1f2, h1f3,h1f4, h1f5, h1fv, h2a, h2ax, h2az, h2b, h2b, h3f2, h3f3b, h3ft, h3t, h4,h4f2, h4f5, h4fa, h4fb, h4fe, h4fg, h4fh, h4fi, h4fj, h4fk, h4fl, h4fm,h4m, h6, ha2, habp1, hadha, hadhb, hadhsc, haf, hagh, hah1, haip1, ha1,hap, hap1, hap2, hars, has2, hat1, hausp, hb1, hb1, hb6, hba1, hba2,hbac, hbb, hbbc, hbd, hbe1, hbegf, hbf2, hbg1, hbg2, hbgr, hbhr, hbm,hbp, hbq1, hbz, hc2, hc3, hca, hcat2, hccs, hcdh, hcf2, hcfc1, hcg, hck,h11, hc12, hc13, hc1s1, hcp, hcp1, hcs, hcvs, hd, hdac1, hdc, hdgf,hdhc7, hdlbp, hdld, hdldt1, hdr, hed, hed, hegf1, hek, hek3, heln1,hem1, hema, hemb, hemc, hempas, hen1, hen2, hep, hep10, her2, her4,herg, herv1, hes1, hesx1, het, hexa, hexb, hf1, hf10, hfc1, hfe, hfe2,hfh11, hfsp, hgd, hgf, hgf, hgf1, hg1, hh, hh72, hhc1, hhc2, hhd, hhh,hhmjg, hhr23a, hht1, hht2, hiap2, higm1, hilda, hint, hiomt, hip, hip1,hip116, hip2, hir, hira, his1, his2, hive1, hivep1, hivep2, hjcd, hk1,hk2, hk3, hk33, hke4, hke6, hkr1, hkr2, hkr3, hkr4, h111, h119, hla-a,hla-b, hla-c, hla-cda12, hla-dma, hia-dmb, hla-dna, hla-dob,hla-dpalhla-dpb1, hla-dqa1, hla-drlb, hla-dra, hla-e, hla-f, hla-g,hla-ha2, hladp, hlaf, hlals, hlcs, hlm2, hlp, hlp3, hlr1, hlr2, hlt,hlx1, hlxb9, hmaa, hmab, hmat1, hmbs, hmcs, hmg1, hmg14, hmg17, hmg2,hmgc1, hmgcr, hmgcs1, hmgcs2, hmgic, hmgiy, hmgx, hmmr, hmn2, hmox1,hmox2, hmr, hms1, hmsn1, hmx1, hmx2, hnd, hnf1a, hnf2, hnf3a, hnf3b,hnf4a, hnp36, hnpcc6, hnrpa1, hnrpa2b1, hnrpd, hnrpf, hnrpg, hnrph1,hnrph2, hnrph3, hnrpk, homg, hops, hox10, hox11, hox12, hox1, hox1a,hox1b, hox1c, hox1d, hox1e, hox1f, hox1g, hox1h, hox1i, hox1j, hox2,hox2a, hox2b, hox2c, hox2d, hox2e, hox2f, hox2g, hox2h, hox21, hox3,hox3a, hox3b, hox3c, hox3d, hox3e, hox3f, hox3g, hox4, hox4a, hox4b,hox4c, hox4d, hox4e, hox4f, hox4g, hox4h, hox4i, hox7, hox8, hoxa1,hoxa10, hoxa11, hoxa13, hoxa3, hoxa4, hoxa5, hoxa6, hoxa7, hoxa9, hoxa,hoxb1, hoxb2, hoxb3, hoxb4, hoxb5, hoxb6, hoxb7, hoxb8, hoxb9, hoxb,hoxc12, hoxc13, hoxc4, hoxc5, hoxc6, hoxc8, hoxc9, hoxc, hoxd1, hoxd10,hoxd11, hoxd12, hoxd13, hoxd3, hoxd4, hoxd8, hoxd9, hoxd, hoxhb9, hp,hp4, hpafp, hpc1, hpc2, hpca, hpca11, hpcx, hpd, hpdr1, hpdr2, hpe1,hpe2, hpe3, hpe4, hpe5, hpect1, hpfh, hpfh2, hpgd, hplh1, hplh2, hpn,hpr, hprt, hprt1, hps, hpt, hpt1, hptp, hptx, hpv18i1, hpv18i2, hpx, hr,hras, hrb, hrc, hrc1, hrca1, hrd, hres1, hrf, hrg, hrga, hrh1, hrh2,hrmt111, hrpt2, hrx, hrx, hry, hsa11, hsa12, hsan1, hsas1, hscr2, hsd11,hsd11b1, hsd11b2, hsd11k, hsd111, hsd17b1, hsd17b2, hsd17b3, hsd17b4,hsd3b1, hsd3b2, hsh, hsn1, hsorc1, hsp27, hsp73, hspa1a, hspa1b, hspa11,hspa2, hspa3, hspa4, hspa5, hspa6, hspa7, hspa8, hspa9, hspb1, hspb2,hspc2, hspca11, hspca12, hspca13, hspca14, hspcb, hspg1, hspg2, hsr1,hsst, hstd, hstf1, htc2, htf4, htk, htk1, ht1, ht1f, htlvr, htn1, htn2,htn3, htnb, htor, htr1a, htr1b, htr1d, htr1e, htr1e1, htr1f, htr2a,htr2b, htr2c, htr3, htr4, htr5a, htr6, htr7, htrx1, hts1, htt, htx,htx1, hub, hud, hup2, hur, hus, hvls, hvbs1, hvbs6, hvbs7, hvem, hvh2,hvh3, hvh8, hxb, hxb1, hy, hya, hya11, hyd2, hygn1, hy1, hyp, hyplip1,hypp, hypx, hyr, hyrc1, hys, ia1, ia2, iap, iapp, iar, iars, ibd1, ibd2,ibm2, ibsp, ica1, icam1, icam2, icam3, icca, ich1, icr2, icr2b, ics1,id1, id2, id3, id4, ida, idd, iddm1, iddm10, iddm11, iddm12, iddm13,iddm15, iddm17, iddm2, iddm3, iddm4, iddm5, iddm6, iddm7, iddm8, iddmx,ide, idg2, idh1, idh2, idh3a, idh3g, ido, ids, idua, ier1, ier3, iex1,if, ifcr, ifgr2, ifi16, ifi27, ifi35, ifi4, ifi5111, ifi54, ifi56,ifi616, ifi78, ifna1, ifna10, ifna13, ifna14, ifna16, ifna17, ifna21,ifna6, ifna7, ifna8, ifna, ifnai1, ifnar1, ifnar2, ifnb1, ifnb2, ifnb3,ifng, ifngr1, ifngr2, ifngt1, ifnr, ifnw1, ifrd2, iga, igat, igb, igbp1,igd1, igda1, igdc1, igds2, iger, iges, igf1, igf1r, igf2, igf2r, igfbp1,igfbp10, igfbp2, igfbp3, igfbp4, igfbp6, igfbp7, igfr1, igfr2, igfr3,igh, igha1, igha2, ighd, ighdy2, ighe, ighg1, ighg2, ighg3, ighg4, ighj,ighm, ighmbp2, ighr, ighv, igi, igj, igk, igkc, igkde1, igkj, igkjrb1,igkv, ig1c, ig1c1, ig1j, ig1p1, ig1p2, ig1v, igm, igo1, igsf1, ihh, ik1,ikba, il10, il10r, il11, il11ra, il12a, il12b, il12rb1, il12rb2, il13,il13ra1, il13ra2, il15, il15ra, il17, il1a, il1b, il1bc, il1r1, il1r2,il1ra, il1rap, il1rb, il1rn, il2, il2r, il2ra, il2rb, il2rg, il3, il3ra,il3ray, il4, il4r, il4ra, il5, il5ra, il6, il6r, il6st, il7, il7r, il8,il8ra, il8rb, il9, il9r, ila, ilf1, il1bp, imd1, imd2, imd4, imd5, imd6,impa1, impdh1, impdh2, impdh11, impg1, impt1, indx, infa2, infa4, infa5,ing1, inha, inhba, inhbb, inhbc, ini1, ink4b, inlu, inp10, inpp1,inpp5a, inpp5b, inpp5d, inpp11, ins, insig1, ins1, ins13, ins14, insr,insrr, int1, int111, int2, int3, int4, int6, iosca, ip2, ipf1, ip1,ipm150, ipox, ipp, ipp2, ipw, iqgap1, ir10, ir20, ireb1, ireb2, irf1,irf2, irf4, irf4, irr, irs1, isa, iscw, is11, is1r, isot, issx, it15,itba1, itba2, itf, itf2, itga1, itga2, itga2b, itga4, itga5, itga6,itga7, itgad, itga1, itgam, itgav, itgax, itgb1, itgb2, itgb3, itgb4,itgb6, itgb7, iti, itih1, itih2, itih3, itih4, itih11, iti1, itk, itm1,itpa, itpka, itpkb, itpr1, itpr2, itpr3, itsn, ivd, iv1, jag1, jak1,jak2, jak3, jbs, jcap, jh8, jip, jk, jme, jmj, joag, jpd, jrk, jrk1,jtk14, jtv1, jun, junb, jund, jup, jv18, jws, k12t, kail, kal1, kar,kars, katp1, kcna1, kcna10, kcna1b, kcna2b, kcna3, kcna4, kcna5, kcna6,kcna7, kcna8, kcna9, kcnab1, kcnab2, kcnb1, kcnc1, kcnc2, kcnc3, kcnc4,kcne1, kcnh1, kcnh2, kcnj1, kcnj10, kcnj11, kcnj12, kcnj15, kcnj3,kcnj4, kcnj5, kcnj6, kcnj6, kcnj7, kcnj8, kcnjn1, kcnk1, kcnk2, kcnk3,kcnma1, kcnq1, kcnq2, kcnq3, kcnq4, kcns2, kd, kdr, ke1, kera, kfl, kfs,kfsd, kfs1, khk, kiaa0122, kid, kid1, kif2, kif3c, kif5b, kip1, kip2,kiss1, kit, klc2, klk1, klk2, klk3, klk3, klkb1, klkr, klrb1, klrc1,klrc2, klrc3, klrc4, klrd1, klst, kms, kms, kng, kno, kns1, kns2, kns11,kns14, kox1, kox11, kox12, kox13, kox15, kox16, kox18, kox19, kox2,kox2, kox22, kox25, kox30, kox32, kox4, kox5, kox6, kox7, kox9, kpna3,kpps1, kpps2, krag, kras1p, kras2, krev1, krg2, krn1, krn11, krox20,krt1, krt10, krt12, krt13, krt14, krt15, krt16, krt17, krt18, krt19,krt2a, krt2e, krt3, krt4, krt5, krt6a, krt6b, krt7, krt8, krt9, krtha2,krtha5, krthb1, krthb6, ks, ktn1, ku70, kup, kvlqt1, kwe, 11.2, 11 cam,123mrp, lab7, lab72, lac, lad, lacs, lad, lad, lad1, laf4, lag3, lag5,lair1, lak1, lalba, lal1, lam1, lama1, lama2, lama3, lama3, lama4,lama5, lamb1, lamb2, lamb2, lamb2t, lamb3, lambr, lamc1, lamc2, lamm,lamnb2, lamp, lamp1, lamp2, lamr1, lams, lap, lap18, laptm5, lar, lar1,lard, large, tars, lbp, lbr, lca, lca1, lcad, lcamb, lcat, lccs, lcfs2,lch, lck, lcn1, lcn2, lco, lcp1, lcp2, lct, ld, ld78, ldb1, ldb2, ldc,ldh1, ldh3, ldha, ldhb, ldhc, ldlr, le, lect2, lef1, lefty1, lefty2,lep, lepr, lerk5, lerk8, leu1, leu7, leut, lfa1a, lfa3, lfh11, lfp,lga1s1, lga1s3, lga1s3 bp, lga1s7, lgcr, lgmd1, lgmd1a, lgmd1b, lgmd1c,lgmd1d, lgmd2b, lgmd2c, lgmd2d, lgmd2e, lgmd2f, lgmd2g, lgmd2h, lgs,lgtn, lhb, lhcgr, lhs, lhx1, lhx3, li, li2, lif, lifr, lig1, lig3, lig4,lim1, lim2, limab1, limk1, limpii, lip2, lipa, lipb, lipc, lipd, lipe,lipo, lis1, lis2, lisx, litaf, lkb1, lkn1, llg11, lman1, lmn1, lmn2,lmna, lmnb1, lmnb2, lmo1, lmo2, lmo3, lmo4, lmo5, lmp10, lmp2, lmp7,lmpx, lms, lmx1, lmx1a, lmx1b, lmyc, lnhr, lnrh, locr, loh11cr2a, lor,lot1, lox, lox1, lox11, lpa, lpaab, lpaata, lpap lpc1, lpc2d, lpd1, lph,lpi, lp1, lpna3, lpp, lps, lpsa, lqt1, lqt2, lqt3, lqt4, lr3, lre1,lre2, lrp, lrp1, lrp2, lrp5, lrp7, lrp8, lrpap1, lrpr1, lrs1, lsamp,lsirf, ls1, lsn, Isp1, lss, lst1, lta, lta4h, ltb, ltb4r, ltbp1, ltbp2,ltbp2, ltbp3, ltbp3, ltbr, ltc4s, Itf, ltk, ltn, lu, lum, luxs, luzp,lw, ly64, ly6e, ly9, lyam1, lyb2, lyf1, ly11, lyn, lyp, lyst, lyt10,lyz, lztr1, m11s1, m130, m17s1, m17s2, m195, m1s1, m3s1, m4s1, m6a, m6b,m6p2, m6pr, m6s1, m7v1, m7vs1, mab211, mac1a, mac2, mac25, macam1, macs,mad, mad211, madd, madh1, madh2, madh3, madh4, madh5, madh6, madh6,madh7, madh9, madm, madr1, maf, mafd1, mafd2, mag, mage1, mageb3,mageb4, mage11, magoh, magp, magp1, magp2, mak, ma1, ma11, man2a2,mana1, mana2, mana2x, manb, manb1, manba, maoa, maob, map1a, map1a1c3,map1b, map1b1c3, map2, map4, map80, map97, mapk1, mapkap3, mapkkk4,mapt, mar, mark3, mars, mas1, masp1, mat1a, mat2a, mata1, mata2, matk,matn1, matn3, max, maz, mb, mbd1, mb1, mb12, mbp, mbp1, mbs, mbs2, mc1r,mc2r, mc3r, mc4r, mc5r, mcad, mcc, mcdc1, mcdr1, mcf2, mcf3, mcfd1,mch2, mch3, mch4, mch5, mckd, mc1, mc11, mcm, mcm2, mcm2, mcm3, mcm6,mcm7, mcmt, mcop, mcor, mcp, mcp1, mcp3, mcph1, mcr, mcs, mcsf, mcsp,mct1, md1, mdb, mdc, mdcr, mddc, mdeg, mdf1, mdg, mdg1, mdh1, mdh2, mdk,mdk, mdm2, mdm4, mdr1, mdr3, mdrs1, mdrv, mds, mds1, mdu1, mdu2, mdu3,mdx, me1, me2, mea, mea6, mec11, mecp2, med, mef, mef2a, mef2b, mef2c,mef2d, mefv, mehmo, meis1, meis2, mekk, mekk1, mekk4, me1, me118, melf,memo1, men1, men2a, meox1, meox2, mep1a, mep1b, mer2, mer6, mest, met,metrs, mfap1, mfap2, mfap3, mfap4, mfd1, mfi2, mfs1, mfs2, mft, mfts,mg50, mga, mga1, mga3, mgat1, mgat2, mgat5, mgc1, mgcn, mgcr, mgct,mgdf, mgea, mgf, mgi, mgmt, mgp, mgsa, mgst1, mgst2, mhc, mhc2ta, mhp2,mhs, mhs2, mhs3, mhs4, mhs6, mia, mic10, mic11, mic12, mic17, mic18,mic2, mic2x, mic2y, mic3, mic4, mic7, mica, micb, mid1, midas, mif, mif,mig, mip, mip2a, mip2b, mip3b, mipep, miff, miwc, mjd, mk, mki67, mkks,mkp2, mkp3, mkpx, mks, mks, mks1, mks2, mla1, mlck, mlf1, mlf2, mlh1,mlk1, mlk3, ml1, ml12, ml1t1, ml1t2, ml1t3, ml1t4, ml1t6, ml1t7, mlm,mlm, mln, mlp, mlr, mlrg, mlrw, mls, mltn, mlvar, mlvi2, mlvt, mmac1,mme, mmp1, mmp10, mmp11, mmp12, mmp13, mmp14, mmp15, mmp16, mmp17,mmp19, mmp2, mmp21, mmp22, mmp3, mmp7, mmp8, mmp9, mn, mn, mnb, mnbh,mnda, mng1, mnk, mns, mnt, mocod, mocs1, mocs2, mody1, mody3, mog, mok2,mom1, mos, mot2, mov34, mox1, mox2, mox44, moz, mp19, mpb1, mpd1, mpdz,mpe, mpe16, mpg, mpi, mpif2, mp1, mp11g, mpo, mpp1, mpp2, mpp3, mppb,mpri, mprn, mps2, mps3a, mps3c, mps4a, mpsh, mpts, mpv17, mpz, mr1,mr77, mrbc, mrc1, mre11, mre11a, mrg1, mrgh, mros, mrp, mrp, mrp1,mrp123, mrs, mrsd, mrsr, mrst, mrx1, mrx14, mrx2, mrx20, mrx21, mrx23,mrx29, mrx41, mrx48, mrx49, mrx9, mrxa, mrxs1, mrxs2, mrxs3, mrxs4,mrxs5, mrxs6, mrxs8, ms3315, ms336, msg1, msh2, msh3, msh4, msh6, msi1,msk16, msk39, msk41, mslr1, msmb, msn, msr1, mss1, mss4, mss4, msse,mst, mst1, mst1r, mstd, mstn, msud1, msx1, msx2, mt1a, mt1b, mt1e, mt1f,mt1g, mt1h, mt1i, mt1j, mt1k, mt1l, mt1x, mt2, mt2a, mt3, mtacr1, mtap,mtbt1, mtcp1, mterf, mtf1, mth1, mthfc, mthfd, mthfr, mtk1, mtm1, mtmr1,mtmx, mtnr1a, mtnr1b, mtp, mtpa, mtr, mtrns, mtrr, mts, mts, mts1, mts1,mts2, mttf1, mtx, mtxn, mu, muc1, muc2, muc3, muc4, muc5, muc5ac, muc5b,muc6, muc8, mu1, mum1, mupp1, musk, mut, mvk, mvlk, mvwf, mwfe, mx, mx1,mx2, mxi1, mxs1, myb, myb11, myb12, mybpc1, mybpc2, mybpc3, mybpcf,mybph, myc, myc11, myc12, myclk1, mycn, myd88, myf3, myf4, myf5, myf6,myh1, myh10, myh11, myh12, myh2, myh3, myh4, myh6, myh7, myh8, myh9,myk1, my1, my11, my12, my13, my14, my15, mylk, mymy, myo10, myo15,myo1a, myo1c, myo1d, myo1e, myo5a, myo6, myo7a, myo9b, myoc, myod1,myog, myp1, myp2, myp3, myr5, mzf1, n33, nab1, nab2, nabc1, nac1a, naca,nacae, nacp, nadmr, naga, nagc, nag1u, nagr1, naip, namsd, nanta3,nap114, nap2, nap21, napb, naptb, nars, nat1, nat1, nat2, nb, nb4s,nbat, nbc3, nbccs, nbccs, nbia1, nbs, nbs, nbs1, nca, ncad, ncam1, ncan,ncbp, ncc1, ncc2, ncc3, ncc4, ncct, ncf1, ncf2, ncf4, nck, nc1, ncst2,ncx1, ncx2, nd, ndhii, ndn, ndp, ndst1, ndufa1, ndufa2, ndufa5, ndufa6,ndufa7, ndufb8, ndufb9, ndufs1, ndufs2, ndufs4, ndufs7, ndufs8, ndufv1,ndufv2, ndufv3, neb, nec1, nec2, nedd1, nedd2, nedd4, nefh, nef1, negf1,negf2, ne111, neb112, nem1, neo1, nep, net, net1, neu, neu, neud4,neurod, neurod2, neurod3, nf1, nf1a, nf2, nfatc1, nfatc2, nfatp, nfe1,nfe2, nfe211, nfe212, nfe2u, nfia, nfib, nfic, nfix, nfkb1, nfkb2,nfkb3, nfkbia, nfkbi11, nfrkb, nfya, nfyb, nga1, ngbe, ngfb, ngfg,ngfic, ngfr, ng1, ngn, nhbp, nhcp1, nhcp2, nhe1, nhe3, nhe4, nhe5,nhlh1, nhlh2, nhp211, nhs, nid, niddm1, ninj1, nipp1, nipsnap1,nipsnap2, nis, nk1r, nkcc1, nkcc2, nkg2, nkg2a, nkg2c, nkg2e, nkg2f,nkhc, nkna, nknar, nknb, nkrp1a, nks1, nksf2, nktr, nkx2a, nkc3.2,nkx3a, nkx6a, nli, nm, nm1, nm23, nmb, nmbr, nmdar1, nmdar2a, nmdar2b,nmdar2c, nmdar2d, nmdara1, nme1, nme2, nme4, nmor1, nmor2, nms1, nmyc,nnat, nmnt, nno1, nog, nol1, nos1, nos2a, nos2b, nos2c, nos3, not,notch1, notch2, notch3, notch4, nov, nov, nov2, nova1, nova3, novp, np,np10, npat, npc, npc1, npd, nph1, nph2, nphl2, nphn, nphp1, nphp2,nphs1, npm1, nppa, nppb, nppc, npps, npr1, npr2, npr3, nps1, npt1, npt2,nptx2, npy, npy1r, npy2r, npy3r, npy5r, npy6r, nqo2, nramp, nramp1,nramp2, nrap, nras, nrb54, nrcam, nrd1, nrf1, nrf1, nrf2, nrgn, nrip1,nrk2, nr1, nrtn, nru, ns1, nsf, nsp, nsp11, nsrd9, nt4, nt5, nt5, ntcp1,ntcp2, ntf3, ntf4, ntf5, nth11, ntn, ntn, ntn21, ntrk1, ntrk2, ntrk3,ntrk4, ntrkr1, ntrkr3, nts, ntt, ntt, nuc1, nucb1, numa1, nup214, nup98,nurr1, nv1, nys1, nys2, nysa, oa1, oa2, oa3, oar, oasd, oat, oat11,oat22, oat23, oatp, oaz, ob, ob10, obf1, obp, obr, oca2, ocm, ocp2,ocr1, ocr11, oct, oct1, oct1, oct2, oct2, oct3, oct7, octn2, octs3,odc1, oddd, odf1, odg1, odod, ofc1, ofc2, ofc3, ofd1, ofe og22, ogdh,ogg1, ogr1, ogs1, ogs2, ohds, ohs, oias, oip1, ok, olf1, olfmf, olfr1,olfr2, omg, omgp, omp, on, op2, opa1, opa2, opa3, opca3, opcm1, opd1,opg1, ophn1, op11, opn, oppg, oprd1, oprk1, oprm1, oprt, opta2, optb1,oqt1, orld2, orlf1, orc11, orc21, orc41, orc51, orfx, orm1, orm2, orw,osbp, osm, osp, ost, ost48, osx, otc, otf1, otf2, otf3, otm, otof, ots,otx1, otx2, ovc, ovcs, ovo11, ox40, oxa11, oxct, oxt, oxtr, ozf, p, p,p1, p15, p16, p167, p28, p2rx3, p2rx4, p2ry1, p2ry2, p2ry4, p2ry7, p2u,p2x3, p2x4, p2y1, p2y2, p2y2, p2y4, p3p40phox, p450c11, p450c17,p450c2a, p450c2d, p450c2e, p450scc, p4ha, p4ha1, p4ha1, p4hb, p5cdh,p79r, pa2g4, pab1, pab2, pabp2, pabp11, pac1, pac1, pacapr, pace, pace4,paep, paf1, paf2, pafah, pafah1b1, pafah1b2, pafah1b3, paga, pah, pahx,pai1, pai2, paics, pak1, pak3, palb, pals, pam, pang, pap, papa, papa2,pappa, par1, par1, par2, par3, par4, par4, par5, park1, park2, park3,pawr, pax1, pax2, pax3, pax4, pax5, pax6, pax7, pax8, pax9, pbca, pbcra,pbfe, pbg pbt, pbx1, pbx2, pbx3, pc, pc1, pc2, pc3, pc3, pca1, pcad,pcap, pcar1, pcbc, pcbd, pcbp1, pcbp2, pcca, pccb, pcdh7, pcdx, pchc,pchc1, pci, pck1, pc1, pc1p, pcm1, pcm1, pcmt1, pcna, pcnt, pcolce, pcp,pcp4, pcs, pctk1, pcsk2, pcsk3, pcsk4, pcsk5, pcsk6, pctk1, pctk3,pcyt1, pdb, pdb2, pdc, pdc, pdcd1, pdcd2, pddr, pde1a, pde1b, pde1b1,pde3b, pde4a, pde4b, pde4c, pde4d, pde5a, pde6a, pde6b, pde6c, pde6d,pde6g, pde6h, pde7a, pdea, pdea2, pdeb pdeb, pdeg, pdeslb, pdgb, pdgfa,pdgfb, pdgfr, pdgfra, pdgfrb, pdha1, pdha2, pdhb, pdj, pdk4, pdnp1,pdnp2, pdnp3, pdr, pds, pds1, pdx1, pdyn, pe1, pea15, pebp2a1, pebp2a3,pecam1, ped, ped, pedf, pee, peg1, peg3, pemp, penk, pent, peo, peo1,peo2, pepa, pepb, pepc, pepd, pepe, pepn, peps, per, per2, peta3, pets1,pex1, pex5, pex6, pex7, pf4, pf4v1, pfas, pfbi, pfc, pfd, pfhb1, pfic1,pfic2, pfkfb1, pfkfb2, pfk1, pfk-mn, pfkp, pfkx, pf1, pfm, pfn1, pfn2,pfrx, pga3, pga4, pga5, pgam1, pgam2, pgamm, pgc, pgd, pgf, pgft, pgk1,pgk2, pgka, pg1, pg11, pg12, pgm1, pgm2, pgm3, pgm5, pgn, pgp, pgp1,pgr, pgs, pgt, pgy1, pgy3, pha1, pha2, pha2a, pha2b, phap1, phb, phc,phe1a, phe3, phex, phf1, phhi, phhi, phk, phka1, phka2, phkb, phkd,phkg1, phkg2, ph1, phl11, phog, phox1, phox2a, php, php1b, phpx, phyh,pi, pi10, pi3, pi4, pi5, pi6, pi7, pi8, pi9, piga, pigc, pigf, pigh,pigr, pik3c2b, pik3ca, pik3r1, pik4cb, pi1, pim1, pin, pin1, pin11, pip,pip5k1b, pir1, pir51, pit, pit1, pitpn, pitx1, pitx2, pitx3, pjs, pk1,pk120, pk3, pk428, pkca, pkcb, pkcc, pkcg, pkcs1, pkd1, pkd2, pkd4,pkdts, pkhd1, pk1r, pkm2, pkp1, pks1, pks1, pks2, pku1, pl, pla2, pla2a,pla2b, pla2g1b, pla2g2a, pla2g4, pla2g4a, pla2g5, pla21, pla21, plag1,plag11, planh1, planh2, planh3, plat, plau, plaur, plb, plc, plc1,plcb3, plcb4, plcd1, plce, plcg1, plcg2, plc1, pld1, plec1, plg, plgf,plg1, pli, pln, plod, plod2, plos1, plp, pls, pls1, plt1, pltn, pltp,plzf, pmca1, pmca2, pmca3, pmca4, pmch, pmch11, pmch12, pmd, pme117,pmi1, pm1, pmm1, pmm2, pmp2, pmp22, pmp35, pmp69, pmp70, pms1, pms2,pms11, pms12, pmx1, pn1, pnd, pnem, pnkd, pnlip, pnmt, pnoc, pod1,podx1, pof, pof1, pol2rb, pola, polb, pold1, pold2, pole, polg, polr2a,polr2c, polr2e, polr2g, polr2i, polrmt, polz, pomc, pon, pon1, pon2,pon3, por, pore, potx, pou1f1, pou2af1, pou3f1, pou3f2, pou3f3, pou3f4,pou4f1, pou4f3, pou5f1, pp, pp14, pp2, pp4, pp5, ppac, ppard, pparg,pparg1, pparg2, ppat, ppbp, ppcd, ppd, ppef1, ppef2, ppfia3, ppgb, pph,pph1, ppia, ppid, ppi11, ppkb, ppks1, ppks2, pp1, pp1a2, ppmx, ppnd,ppnoc, ppo1, ppox, ppp1a, ppp1ca, ppp1cb, ppp1cc, ppp1r2, ppp1r5,ppp1r7, pppd1r8, ppp2b, ppp2ca, ppp2cb, ppp2r1b, ppp2r4, ppp2r5a,ppp2r5b, ppp2r5c, ppp2r5d, ppp2r5e, ppp3ca, ppp3cb, ppp3cc, ppp3r1,ppp4c, ppp5c, ppt, ppt2, ppx, ppy, ppyr1, pr, prad1, prb1, prb2, prb3,prb4, prca1, prca2, prcc, prcp, pre1p, prep, prf1, prg, prg1, prg1,prgs, prh1, prh2, prim1, prim2a, prim2b, prip, prk1, prkaa1, prkaa2,prkab1, prkaca, prkacb, prkacg, prkag1, prkag2, prkar1a, prkar1b,prkar2b, prkca, prkcb1, prkcd, prkcg, prkci, prkcl1, prkcnh1, prkcq,prkcsh, prkdc, prkg1, prkg1b, prkg2, prkgr1b, prkgr2, prkm1, prkm3,prkm4, prkm9, prkn, prkr, prkx, prky, pr1, pr1r, prm1, prm2, prmt2,prnp, proa, proc, prodh, prohb, prop1, pros1, pros30, prox1, prp8, prph,prps1, prps2, pipsap1, prr1, prr2, prs, prsc1, prss1, prss11, prss2,prss7, prss8, prss11, prtn3, prts, psa, psa, psach, psap, psbg1, psbg2,psc2, psc5, psca, psd, psen1, psen2, psf1, psf2, psg1, psg11, psg12,psg13, psg2, psg3, psg4, psg5, psg6, psg7, psg8, psg11, pskh1, psm,psma1, psma2, psma3, psma5, psmb1, psmb10, psmb2, psmb3, psmb4, psmb5,psmb8, psmb9, psmc1, psmc2, psmc3, psmc5, psmd7, psmd9, psme1, psme2,psors1, psors2, psors3, psp, psps1, psps2, pss1, psst, pst, pst, pst1,psti, ptafr, ptc, ptc, ptc, ptch, ptd, pten, ptgds, ptger1, ptger2,ptger3, ptgfr, ptgfrn, ptgir, ptgs1, ptgs2, pth, pthlh, pthr, pthr1,pthr2, ptk1, ptk2, ptk2b, ptk3, ptk7, ptlah, ptma, ptms, ptn, ptos1,ptp18, ptp1b, ptp4a1, ptp4a2, ptpa, ptpa, ptpd, ptpg, ptpg1, ptpgmc1,ptpn1, ptpn10, ptpn11, ptpn12, ptpn13, ptpn14, ptpn2, ptpn5, ptpn6,ptpn7, ptpra, ptprb, ptprc, ptprcap, ptprd, ptpre, ptprf, ptprg, ptprh,ptprj, ptprk, ptpr11, ptpr12, ptprm, ptprn, ptpro, ptprs, ptprz1, ptpt,pts, pts1r, ptx1, ptx3, pujo, pum, pur1, pur1, pura, pvalb, pvr, pvr11,pvr12, pvrr1, pvrr2, pvs, pvt1, pwcr, pwp2, pwp2h, pws, pxaaa1, pxe,pxe1, pxf, pxmp1, pxmp11, pxmp3, pxr1, pycr1, pycs, pygb, pyg1, pygm,pyk2, pyst1, pyst2, pzp, gars, qdpr, qin, qm, qpc, qprs, rab, rab1,rab13, rab1a, rab21, rab3a, rab3b, rab4, rab5, rab5a, rab6, rab7,rabgdla, rabgdib, rabggta, rabggtb, rabif, rac2, rac3, rad1, rad17,rad23a, rad23b, rad51a, rad51c, rad51d, rad5311, rad52, rad54, rad6a,rad6b, raf1, rafa1, rag1, rag2, rage, rala, ralb, ralgds, ramp,ranbp211, ranbp3, rao, rap1a, rap1b, rap1ga1, rap1gds1, rap2a, rap74,rapsn, rara, rarb, rarg, rars, rasa1, rasa2, rasgfr3, rask2, rb1, rbbp2,rbbp5, rbbp6, rb11, rb12, rbm1, rbm2, rbm3, rbmy1a1, rbp1, rbp2, rbp3,rbp4, rbp5, rbp56, rbp6, rbq3, rbtn1, rbtn11, rbtn12, rca1, rcac, rcc1,rccp1, rccp2, rcd1, rcd2, rcdp1, rcn1, rcn2, rcp, rcv1, rd, rdbp, rdc7,rdp, rdpa, rdrc, rds, rdt, rdx, reca, recc1, recq1, red1, red2, reg,reg1a, reg1, re1, re1a, re1n, ren, renbp, rens1, rent1, rep8, req, ret,rev3, rev31, rfc1, rfc2, rfc3, rfc4, rfc5, rfp, rfx1, rfx2, rfx5,rfxank, rfxap, rgc1, rgr, rgs, rgs1, rgs14, rgs16, rgs2, rgs2, rgs3,rgs5, rh50a, rhag, rhbd1, rhc, rhce, rhd, rheb2, rho, rho7, rhogap2,rhogap3, rhoh12, rhoh6, rhoh9, rhok, rhom1, rhom2, rhom3, rieg1, rieg2,rige, rigui, ring1, ring10, ring11, ring12, ring3, ring31, ring4, ring5,ring6, ring7, rip, rip140, riz, rk, rl, rlbp1, rlf, rln1, rln2, rmch1,rmd1, rmrp, rmrpr, m5s1, rnase1, rnase2, rnase3, rnase4, rnase5, rnase6,rnase1, rnase1i, rne1, rnf1, rnf3, rnf4, rnf5, rnh, rnpep, rnpulz, rnr1,rnr2, rnr3, rnr4, rnr5, rns1, rns2, rns3, rns4, rns4, rns4i, rntmi,rnu1, rnu15a, rnu17a, rnu17b, rnu1a, rnu2, rnu3, ro52, rom1, romk1, ron,ror1, rora, rorb, rorc, rorg, ros1, rosp1, rox, rp1, rp10, rp105, rp11,rp12, rp13, rp14, rp15, rp17, rp18, rp19, rp2, rp22, rp24, rp25, rp3,rp4, rp6, rp7, rp9, rpa1, rpa2, rpa3, rpd311, rpe, rpe65, rpe119rp122,rp123a, rp1231, rp129, rp130, rp135a, rp136a, rp17a, rpms12, rpn1, rpn2,rpo12, rps11, rps14, rps17, rps17a, rps17b, rps1711, rps1712, rps18,rps20a, rps20b, rps24, rps25, rps3, rps4x, rps4y, rps6, rps6ka1,rps6ka2, rps6ka3, rps8, rpsm12, rptpm, rpu1, rpx, rrad, rras, rrbp1,rreb1, rrm1, rrm2, rrp, rrp22, rs1, rs1, rsc1a1, rsk1, rsk2, rsk3, rsn,rss, rsts, rsu1, rt6, rtef1, rtkn, rtn1, rtn2, rts, rts, rtt, rws, rxra,rxrb, rxrg, ryr1, ryr2, ryr3, rzrb, rzrg, s100a1, s100a10, s100a11,s100a12, s100a13, s100a2, s100a3, s100a4, s100a5, s100a6, s100a7,s100a8, s100a9, s100b, s100d, s100e, s100, s100p, s152, s4, s7, saa1,saa2, saa4, sacs, safb, sag, sah, sahh, sai1, sakap84, sal11, sal12,sams1, sams2, sap, sap1, sap1, sap2, sap62, sar, sar1, sar2, sard, sas,sat, satb1, satt, sbma, sc, sc1, sc5d1, sca1, sca10, sca2, sca2, sca3,sca4, sca5, sca6, sca7, sca8, sca8, scar, scca1, scca2, sccd, scd, sceh,scg1, scg2, scg3, schad, scida, scidx, scidx1, sc1, sc1c1, sc11, scn,scn1a, scn1b, scn2a, scn2a1, scn2a2, scn2b, scn3a, scn4a, scn5a, scn6a,scn8a, scnn1a, scnn1b, scnn1d, scnn1g, scot, scp, scp1, scp2, scpn,scra1, scra1, scs, sctr, scya1, scya11, scya13, scya14, scya15, scya16,scya19, scya2, scya21, scya22, scya24, scya25, scya3, scya311, scya4,scya5, scya7, scya8, scyb5, scyb6, scyd1, sczd1, sczd2, sczd3, sczd4,sczd5, sczd6, sczd7, sczd8, sdc1, sdc2, sdc4, sdf1, sdf2, sdh1, sdh2,sdha, sdhb, sdhc, sdhd, sdhf, sds22, sdty3, sdys, se, sea, sec1311,sec13r, sec141, sec7, sed1, sedt, sef2, sel11, sele, sell, selp, selp1g,sema3f, sema4, sema5, semg, semg1, semg2, sen1, sep, sepp1, serca1,serca3, serk1, ses1, set, sex, sf, sf1, sfa1, sfd, sfmd, sfrs1, sfrs2,sfrs7, sftb3, sftp1, sftp2, sftp4, sftpa1, sftpa2, sftpb, sftpc, sftpd,sgb, sgca, sgcb, sgcd, sgcg, sgd, sgk, sglt1, sglt2, sgm1, sgne1, sgp2,sgpa, sgsh, sh2d1a, sh3 bp2, sh3d1a, sh3gbr, sh3p17, shb, shbg, shc1,shc11, shfd1, shfd2, shfm1, shfm2, shfm3, shh, ship, shmt1, shmt2,shoc2, shot, shox, shox2, shps1, shs, shsf1, si, siah1, siah2, siasd,siat1, siat4, siat4c, siat8, sids, si1, si1v, sim1, sim2, sipa1, sis,siv, six1, six5, sja, sjs, ski, ski2, ski2w, skiv21, skp1a, skp1b, skp2,sla, slap, slbp, slc, slc10a1, slc10a2, slc12a1, slc12a2, slc12a3,slc14a1, slc14a2, slc15a1, slc16a1, slc16a2, slc17a1, slc17a2, slc18a1,slc18a2, slc18a3, slc19a1, slc1a1, slc1a2, slc1a3, slc1a4, slc1a5,slc20a1, slc20a2, slc20a3, slc21a2, slc21a3, slc22a1, slc22a2, slc22a5,slc2a1, slc2a2, slc2a3, slc2a4, slc2a5, slc2c, slc3a1, slc4a1, slc4a2,slc4a6, slc5a1, slc5a2, slc5a3, slc5a5, slc6a1, slc6a10, slc6a12,slc6a2, slc6a3, slc6a4, slc6a6, slc6a8, slc6a9, slc7a1, slc7a2, slc7a4,slc7a5, slc7a7, slc8a1, slc8a2, slc9a1, slc9a2, slc9a3, slc9a4, slc9a5,sld, sle1, sleb1, slim1, stn, slo, slos, slp76, sls, slug, sm1, sm22,sma4, smad1, smad1, smad2, smad3, smad4, smad5, smad6, smad7, smad9,sma1, smam1, smarca1, smarca2, smarca3, smarca5, smarcb1, smax2, smc1,smcc, smcr, smex, smcy, sml1, smn, smn1, smn2, smnr, smo, smoh, smpd1,sms, smt3, smt3h1, smtn, smubp2, sn, snap25, snat, snca, sncb, sncg,snf2h, snf211, snf212, snf213, snf5, sn1, snn, snrp70, snrpa, snrpe,snrpn, snt1, snt2b1, snt2b2, sntb1, snt1, snx, soat, sod1, sod2, sod3,solh, son, sord, sor11, sos1, sos2, sox1, sox10, sox11, sox2, sox20,sox22, sox3, sox4, sox9, sp1, sp1, sp3, sp3, sp4, spa1, spag1, spag4,spam1, sparc, spat, spbp, spch1, spd, spf30, spg3a, spg4, spg5a, spg6,spg7, spg8, spg9, spgp, spgy1a, sph2, spi1, spink1, spk, spmd, spn,spp1, spp2, sppm, spr, sprk, sprr1a, sprr1b, sprr2a, sprr2b, sprr2c,sprr3, sps1, spsma, spta1, sptan1, sptb, sptbn1, sra1, sra2, src, src1,src1, src2, srd5a1, srd5a2, srebf1, srebf2, sri, srk, srm, srn1, srp14,srp19, srp46, srpr, srpx, srs, srvx, sry, ss, ss, ssa, ssa1, ssa2,ssadh, ssav1, ssbp, ssdd, ssr2, ssrc, sst, sstr1, sstr2, sstr3, sstr4,sstr5, ssx1, ssxt, st2, st3, st4, st5, st6, st8, sta, stac, stam, star,stat, stat1, stat3, stat4, stat5, ssx1, stc1, stch, std, std, step,step, stf1, stfa, stfb, stgd1, stgd2, stgd3, stgd4, sthe, stk1, stk11,stk15, stk2, stk6, sti, stm, stm2, stm7, stmy1, stmy2, stmy3, stp, stp1,stp2, sts, sts1, stx, stx1b, stx7, stxbp1, stxbp2, sultlc1, supt6h, sur,sur1, surf1, surf2, surf3, surf4, surf5, surf6, svct2, svmt, sw, sxi2,syb1, syb2, syb11, sycp1, syk, sym1, syn1, syn2, syn3, syngap, syns1,syp, syt, syt1, syt2, syt3, syt4, syt5, t, t3d, taa16, tac1r, tac2,tac2r, tac3, tacr1, tacr2, taf2, taf2a, taf2a, taf2d, taf2h, taf2n,tafii100, tagln, tak1, tal1, tal2, taldo1, tam, tan1, tap1, tap2, tapa1,tapbp, tapvr1, tars, tas, task, tat, taut, tax, tax1, taz, tbg, tbp,tbp1, tbs, tbx1, tbx2, tbx3, tbx5, tbxa2r, tbxas1, tc1, tc2, tcbp, tcd,tcea1, tceb11, tceb3, tcf1, tcf12, tcf13, tcf1311, tcf14, tcf15, tcf17,tcf19, tcf2, tcf20, tcf21, tcf3, tcf4, tcf5, tcf611, tcf612, tcf7, tcf8,tcf9, tcfeb, tcf11, tcf14, tcl1, tcl1a, tcl2, tcl3, tcl4, tcl5, tcn1,tcn2, tco, tcof1, tcp1, tcp10, tcp11, tcp228, tcpt, tcra, tcrb, tcrd,tcrg, tcrz, tcs1, tcta, tcte1, tcte3, tcte11, tdf, tdfa, tdfx, tdg,tdgf1, tdn, tdo, tdo2, tdt, tead4, tec, tec, teck, tecta, tef, tegt,tek, te1, tem, tep1, terc, terf1, tert, tes1, tesk1, tex28, tf, tf2s,tf6, tfa, tfam, tfap2a, tfap2b, tfap2c, tfap4, tfcoup1, tfcoup2, tfcp2,tfdp1, tfdp2, tfe3, tff1, tff2, tff3, tfiiia, tfn, tfpi, tfpi2, tfr,tfrc, tfs1, tft, tg, tg737, tgb1, tgb2, tgd, tgfa, tgfb1, tgfb2, tgfb3,tgfb4, tgfbi, tgfbr1, tgfbr2, tgfbr3, tgfbre, tgfr, tgm1, tgm2, tgm3,tgm4, tgn38, tgn46, th, thas, thbd, thbp1, thbs1, thbs2, thbs3, thc,thh, th1, thop1, thpo, thr1, thra, thra1, thra1, thrb, thrm, thrsp,thy1, tial1, tiam1, tiar, tic, tie, tie1, tie2, tigr, ti1, til3, til4,tim, timp, timp1, timp2, timp3, tinur, titf1, titf2, tjp1, tk1, tk2,tkc, tkcr, tkr, tkt, tkt2, tkt11, tla519, tlcn, tle1, tle2, tle3, tlh1,tln, tlr1, tlr2, tlr3, tlr4, tlr5, tm4sf1, tm4sf2, tm7sf2, tmc, tmd,tmdci, tmem1, tmf1, tmip, tmod, tmp, tmpo, tmprss2, tms, tmsa, tmsb,tmvcf, tna, tndm, tnf, tnfa, tnfaip1, tnfaip2, tnfaip4, tnfaip6, tnfar,tnfb, tnfbr, tnfc, tnfcr, tnfr1, tnfr2, tnfrsf10b, tnfrsf12, tnfrsf14,tnfrsf16, tnfrsf17, tnfrsf1a, tnfrsf1b, tnfrsf4, tnfrsf5, tnfrsf6,tnfrsf6b, tnfrsf7, tnfrsf8, tnfrsf9, tnfsf11, tnfsf12, tnfsf5, tnfsf6,tnfsf7, tnnc1, tnnc2, tnni1, tnni2, tnni3, tnnt1, tnnt2, tnnt3, tnp1,tnp2, tnr, tns, tnx, tnxa, toc, top1, top2, top2a, top2b, top3, tp1,tp120, tp250, tp53, tp53 bp2, tp63, tp73, tpa, tpbg, tpc, tpc, tph,tph2, tpi1, tp12, tpm1, tpm2, tpm3, tpm4, tpmt, tpo, tpo, tpp2, tpr,tpr1, tprd, tps1, tps2, tpsn, tpst1, tpst2, tpt, tpt1, tptps, tpx, tpx1,tr, tr2, tr4, tra1, traf1, traf5, trailr2, tran, trance, trap170, trc3,trc8, tre, treb36, trek, trf1, trg1, trh, trhr, tric5, trio, trip1,trip14, trip6, trk, trk1, trka, trkb, trkc, trke, tr11, tr12, trm1,trm1, trm2, trma, trmi1, trmi2, trn, trn1, tro, trp1, trp1, trp2, trp3,trpc1, trpm2, trpo, trps1, trps2, trq1, trr, trr3, trrap, trsp, trt1,trt2, trv1, trv2, trv3, trv4, trv5, try1, try2, ts, ts13, ts546, tsbn51,tsc tsc1, tsc2, tsd, tse1, tsg101, tsg7, tshb, tshr, tsix, tsp3, tspy,tssc3, tst1, tst1, tsta3, tsy, ttc1, ttc3, ttf, ttf1, ttf2, ttg2, ttim1,ttn, ttp, ttp1, ttpa, ttr, tuba3, tubal1, tubb, tufm, tuft1, tulp1,tuple1, tw, tweak, twik1, twist, txgp11, txk, txn, txnr, txnrd1, tyh,tyk1, tyk2, tyk3, tyms, tyr, tyr1, tyro3, tyrp1, tyrp2, tys, u17hg,ulrnp, u22hg, u2af1, u2aflrs1, u2aflrs2, u2aflrs3, uba52, ubb, ubc,ubc4, ubc7, ubc8, ubch2, ubc1, ube1, ube2, ube2a, ube2b, ube2e2, ube2g,ube2g2, ube2h, ube2i, ube211, ube2v1, ube3a, ubh1, ubid4, ub11, uch11,ucn, ucp1, ucp2, ucp3, udpgdh, uev1, ufd11, ufs, ugalt, ugb, ugcg, ugdh,ugn, ugp1, ugp2, ugpp2, ugt1, ugt1a1, ugt2b11, ugt2b15, ugt2b17, ugt2b4,ugt2b7, ugt2b8, ugt2b9, ugt1, uhg, uhx1, ukhc, umod, umph2, umpk, umps,unc18, unc18b, und, ung, unr, unr, uox, up, upk1b, ups, uqbp, uqcrb,uqcrc1, uqcrc2, uqcrfs1, uqor1, uqor13, uqor22, urk, urkr, uroc, urod,uros, usf1, usf2, ush1, ush1a, ush1b, ush1c, ush1d, ush1e, ush1f, ush2a,ush3, usp11, usp5, usp7, usp9x, usp9y, ut1, ut2, ute, utr, utrn, utx,uty, uv20, uv24, uvo, vacht, vacm1, vamp1, vamp2, vars1, vasp, vat1,vat2, vav, vav1, vav2, vbch, vbp1, vcam1, vcf, vcl, vcp, vdac1, vdac2,vdd1, vdi, vdr, vegf, vegfb, vegfd, vegfr3, vgf, vgl, vgr1, vhl, vhr,vil1, vil2, vim, vip, vipr1, vipr2, vis1, vla1, vla5a, vlacs, vlcad,vldlr, vmat1, vmcm, vmd1, vmd2, vnra, vnt, vp, vpp1, vpp3, vpreb1,vpreb2, vrf, vrk1, vrk2, vrnf, vrni, vsn11, vtn, vwf, vws, waf1, wars,was, wbs, wd1, wdr2, wee1, wfrs, wfs, wfs1, wgn1, whcr, wi, wisp1,wisp2, wisp3, wnd, wnt1, wnt10b, wnt13, wnt14, wnt15, wnt2, wnt3, wnt5a,wnt7a, wnt7b, wnt8b, wrb, wrn, ws1, ws2a, ws2b, ws4, wsn, wss, wss, wt1,wt2, wt3, wt4, wt5, wts, wts1, wws, x11, xbp1, xbp2, xce, xdh, xe169,xe7, xe7y, xg, xgr, xh2, xiap, xic, xist, xk, xla, xla2, xlp, xlpd,xlrs1, xm, xpa, xpb, xpc, xpcc, xpct, xpf, xpf, xpg, xpmc2h, xpnpep2,xpo1, xrcc1, xrcc2, xrcc3, xrcc4, xrcc5, xrcc9, xrs, xs, xwnt2, yb1,yes1, yk140, y11, yrrm1, yt, ywha1, ywhab, ywhah, ywhaz, yy1, zac, zag,zan, zap70, zf87, zfm1, zfp3, zfp36, zfp37, zfx, zfy, zic1, zic2, zic3,zipk, znf1, znf10, znf117, znf11a, znf11b, znf12, znf121, znf123,znf124, znf125, znf126, znf13, znf14, znf141, znf144, znf146, znf147,znf157, znf16, znf160, znf162, znf163, znf165, znf169, znf173, znf179,znf189, znf19, znf192, znf193, znf195, znf198, znf2, znf20, znf200,znf204, znf217, znf22, znf23, znf24, znf25, znf26, znf27, znf29, znf3,znf32, znf34, znf35, znf36, znf38, znf4, znf40, znf41, znf42, znf44,znf45, znf46, znf5, znf6, znf69, znf7, znf70, znf71, znf72, znf73,znf74, znf75, znf75a, znf75c, znf76, znf77, znf79, znf8, zn80, znf81,znf83, znf9, znfc150, znfc25, znfxy, znf3, znt4, zp3a, zp3b, zpk, zws1,and zyx.

Furthermore, genes from bacteria, plants, yeast, and mammals (e.g.,mice) can be used with the microorganisms provided herein. Non-limitingexamples of E. coli genes include: aarF, aas, aat, abpS, abs, accA,accB, accC, accD, acd, aceA, aceB, aceE, aceF, aceK, ackA, ackB, acnA,acnB, acpD, acpP, acpS, acpX, acrA, acrB, acrC, acrD, acrE, acrF, acrR,acs, ada, add, adhB, adhC, adhE, adhR, adiA, adiY, adk, aegA, aer, aes,agaA, agaB, agaC, agaD, agaI, agaR, agaS, agaV, agaW, agaZ, agp, ahpC,ahpF, aidB, ais, alaS, alaT, alaU, alaV, alaW, alaX, aldA, aldB, aldH,alkA, alkB, alpA, air, alsA, alsB, alsC, alsE, alsK, alx, amiA, amiB,amn, ampC, ampD, ampE, ampG, ampH, amtB, amyA, ansA, ansB, apaG, apaH,aphA, appA, appB, appC, appY, apt, aqpZ, araA, araB, araC, araD, araE,araF, araG, araH, arai, arcA, arcB, argA, argB, argC, argD, argE, argF,argG, argH, argl, argM, argP, argQ, argR, argS, argT, argU, argV, argW,argX, argY, argZ, aroA, aroB, aroC, aroD, aroE, aroF, aroG, aroH, aroI,aroK, aroL, aroM, aroP, aroT, arsB, arsC, arsR, artl, arts, artM, artP,artQ, ascB, ascF, ascG, asd, aslA, aslB, asmA, asnA, asnB, asnC, asnS,asnT, asnU, asnV, asnW, aspA, aspC, aspS, aspT, aspU, aspV, asr, asu,atoA, atoB, atoC, atoD, atoS, atpA, atpB, atpC, atpD, atpE, atpF, atpG,atpH, atpI, avtA, azaA, azaB, azI, bacA, baeR, baeS, barA, basR, basS,bax, bcp, bcr, betA, betB, betI, betT, bfd, bfm, bfr, bglA, bglB, bglF,bglG, bglJ, bglT, bglX, bioA, bioB, bioC, bioD, bioF, bioH, bioP, bipA,birA, bisC, bisZ, blc, bolA, bRNQ, brnR, brnS brnT, btuB, btuc, btuD,btuE, btuR, bymA, cadA, cadB, cadC, cafA, caiA, caiB, caiC, caiD, caiE,caiF, caiT, calA, caiC, calD, can, carA, carB, cbl, cbpA, cbt, cca,ccmA, ccmB, ccmC, ccmD, ccmE, ccmF, ccmG, ccmH, cdd, cde, cdh, cdsA,cdsS, cedA, celA, celB, celC, celD, celF, cfa, cfcA, chaA, chaB, chaC,cheA, cheB, cheR, cheW, cheY, cheZ, chpA, chpB, chpR, chpS, cirA, citA,citB, cld, cipA, clpB, clpP, clpX, cls, cmk, cmlA, cmr, cmtA, cmtB,coaA, cobS, cobT, cobU, codA, codB, cof, cog?, corA, cpdA, cpdB, cpsA,cpsB, cpsC, cpsD, cpsE, cpsF, cpsG, cpxA, cpxB, cpxP, cpxR, crcA, crcB,creA, creB, creC, creD, crg, crl, crp, crr, csdA, csgA, csgB, csgD,csgE, csgF, csgG, csiA, csiB, csiC, csiD, csiE, csiF, cspA, cspB, cspC,cspD, cspE, cspG, csrA, csrB, cstA, cstC, cup, cutA, cutC, cutE, cutF,cvaA(ColV), cvaB(ColV), cvaC(Co-lV), cvi(ColV), cvpA, cxm, cyaA, cybB,cybC, cycA, cydA, cydB, cydC, cydD, cynR, cynS, cynT, cynX, cyoA, cyoB,cyoC, cyoD, cyoE, cysA, cysB, cysC, cysD, cysE, cysG, cysH, cyst, cyst,cysK, cysM, cysN, cysP, cysQ, cysS, cysT, cysU, cysW, cysX?, cysZ?,cytR, dacA, dacB, dacC, dacD, dadA, dadB, dadQ, dadX, dam, dapA, dapB,dapD, dapE, dapF, dbpA, dcd, dcm, dcp, dcrB, dctA, dctB, dcuA, dcuB,dcuC, ddIA, ddlB, ddpA, ddpB, ddpC, ddpD, ddpF, ddpX, deaD, dedA, dedD,def, degP, degQ, degS, del, deoA, deoB, deoC, deoD, deoR, dfp, dgd,dgkA, dgkR, dgoA, dgoD, dgoK, dgoR, dgoT, dgsA, dgt, dicA, dicB, dicC,dicF, dinB, dinD, dinF, dinG, dint, dinY, dipZ, djiA, dksA, dld, dmsA,dmsB, dmsC, dnaA, dnaB, dnaC, dnaE, dnaG, dnal, dnaJ, dnaK, dnaL, dnaN,dnaQ, dnaT, dnaX, dppA, dppB, dppC, dppD, dppF, dppG, dps, dsbA, dsbB,dsbC, dsbG, dsdA, dsdC, dsdX, dsrA, dsrB, dut, dvl, dxs, ebgA, ebgB,ebgC, ebgR, ecfa, eco, ecpD, eda, edd, efp, enirA, emrB, emrD, emrE,endA, eno, entA, entB, entC, entD, entE, entF, envN envP, envQ, envR,envT, envY, envZ, epd, EppA, minigene, EppB, minigene, EppC, minigene,EppD, minigene, EppE, minigene, EppG, minigene, EppH, minigene, era,esp, evgA, evgS, exbB, exbC, exbD, expA, exuR, exuT, fabA, fabB, fabD,fabF, fabG, fabH, fabl, fabZ, fadA, fadB, fadD, fadE, fadH, fadL, fadR,farR, fatA, fbaA, fbaB, fbp, fcl, fcsA, fdhD, fdhE, fdhF, fdnG, fdnH,fdnl, fdoG, fdoH, fdol, fdrA, fdx, feaB, feaR, fecA, fecB, fecC, fecD,fecE, feel, fecR, feoA, feoB, fepA, fepB, fepC, fepD, fepE, fepG, fes,fexB, ffh, ffs, fhlA, fhlB, fhuA, fhuB, fhuD, fhuE, fhuF, fic, fimA,fimB, fimC, fimD, fimE, fimF, fimG, fimH, fiml, fipB, fipC, fis, fiu,fixA, fixB, fixC, fixX, fklB, fkpA, fldA, flgA, flgB, flgC, flgD, flgE,flgF, flgG, flgH, flgl, flgJ, flgK, flgL, flgM, flgN, flhA, flhB, flhc,flhD, fliA, fliC, fliD, fliE, fliF, fliG, fliH, fliI, fliJ, fliK, fliL,fliM, fliN, fliO, flip, fliQ, fliR, fliS, fliT, fliY, fliZ, flk, flu,fmt, fnr, focA, focB, folA, folC, folD, folE, folK, folP, folX, fpr,frdA, frdB, frdC, frdD, frr, fruA, fruB, fruK, fruR, fsr, ftn, ftsA,ftsE, ftsl, ftsJ, ftsK, ftsL, ftsN, ftsQ, ftsW, ftsX, ftsY, ftsZ, fucA,fuel, fucK, fucO, fucP, fucR, fumA, fumB, fumC, fur, fusA, fusB, gabCgabD, gabP, gabT, gadA, gadB, gadR, galE, galF, galK, galM, galP, gaiR,galS, galT, galU, gapA, gapC, garA, garB, gatA, gatB, gatC, gatD, gatR,gatY, gatZ, gcd, gel, gcpE, gcvA, gcvH, gcvP, gcvR, gcvT, gdhA, gef,ggt, gidA, gidB, gip, glcB, glcC, glcD, gleE, glcG, gldA, glf, glgA,glgB, glgC, glgP, glgS, glgX, glk, glmM, glmS, glmU, glmX, glnA, glnB,glnD, glnE, glnG, glnH, glnK, glhL, glnP, glnQ, glnR, glnS, glnT, glnU,glnV, glnW, glnX, gloA, glpA, glpB, glpC, glpD, gipE, gipF, gipG, glpK,glpQ, gipR, glpT, glpX, gltA, gltB, gltD, gltE, gltF, gltH, gltJ, gltK,gltL, gltM, gltP, gltR, gltS, gltT, gltU, gltv, gltW, gltX, glyA, glyQ,glyS, glyT, giyU, glyv, glyW, glyX, glyY, gmd, gmk, gmm, gnd, gntK,gntP, gntR, gntS, gntT, gntU, gntV, goaG, gor, gph, gpmA, gpp, gprA,gprB, gpsA, gpt, greA, greB, groL, groS, grpE, grxA, grxB, grxC, gshA,gshB, gsk, gsp, gsp*, gst, guaA, guaB, guaC, gurB, gurC, gutM, gutQ,gyrA, gyrB, hcaB, hcaC, hcaD, hcaE, hcaF, hcaR, hcaT, hdeA, hdeB, hdeD,hdhA, helD, hemA, hemB, hemC, hemD, hemE, hemF, hemG, hemH, hemK, hemL,hemM, hemX, hemY, hepA, het, hflB, hflC, hflK, hflX, hfq, hha, hipA,hipB, hisA, hisB, hisC, hisD, hisF, hisG, hisH, hisI, hist, hisM, hisP,hisQ, hisR, hisS, hipA, hlyE, hmp, hns, holA, holB, holC, holD, holE,hopB, hopC, hopD, hpt, hrpA, hrpB, hrsA, hscA, hscB, hsdM, hsdR, hsdS,hslC, hslD, hslE-H, hslJ, hslK, hsIL-N, hslO-R, hslU, hslV, hslW, htgA,htpG, htpX, htrB, htrC, htrE, htrL, hupA, hupB, hyaA, hyaB, hyaC, hyaD,hyaE, hyaF, hybA, hybB, hybC, hybD, hybE, hybF, hybG, hycA, hycB, hycC,hycD, hycE, hycF, hycG, hycH, hycI, hydA, hydG, hydH, hydN, hyfA, hyfB,hyfC, hyfD, hyfE, hyfF, hyfG, hyffl, hyfl, hyfJ, hyfR, hypA, hypB, hypC,hypD, hypE, hypF, iadA, iap, ibpA, ibpB, icd, iclR, ihfA, ihfB, iteR,ileS, ileT, ileU, ileV, ileX, ileY, ilvA, ilvB, ilvC, ilvD, ilvE, ilvF,ilvG, ilvH, ilyl, ilvJ ilvM, ilvN, ilvR, ilvU, ilvY, imp, inaA, inaR,infA, infB, infC, inm, insA(IS1), intA, isb(IS1), isfA, ispA, ispB,KanR, katE, katG, kba, kbl, kch, kdgK, kdgR, kdgT, kdpA, kdpB, kdpC,kdpD, kdpE, kdpF, kdsA, kdsB, kdtA, kdtB, kefB, kefC, kgtp, ksgA, ksgB,ksgC, ksgD, lacA, lacI, lacY, lacZ, lamB, lar, ldcC, ldhA, lepA, lepB,leuA, leuB, leuC, leuD, leuJ, leuO, leuP, leuQ, leuR, leuS, leuT, leuU,leuV, leuW, leuX, leuY, leuZ, lev, lexA, lgt, lhr, ligA, ligT, linB,lipA, lipB, lit, livF, livG, livH, livJ, livK, livM, lldD, lldP, lldR,lolA, lon, lpcA, lpcB, lpd, lplA, lpp, lpxA, lpxB, lpxC, lpxD, lpxK,lrb, lrhA, lrp, Irs lspA, lysA, lysC, lysP, lysQ, lysR, lysS, lysT,lysU, lysV, lysW, lysX, lysY, lysZ, lytA, lytB, lyx, maa, mac, mae,mafA, mafB, malE, malF, malG, malI, malK, malM, malP, malQ, malS, malT,malX, malY, malZ, manA, manC, manX, manY, manZ, map, marA, marB, marR,mbrB, mcrA, mcrB, mcrC, mcrD, mdaB, mdh, mdoB, mdoG, mdoH, meb, melA,melB, meiR, menA, menB, menC, menD, menE, menF, mepA, mesJ, metA, metB,metC, metD, metE, metF, metG, metH, metJ, metK, metL, metR, metT, metU,metV, metW, metY, metZ, mfd, mglA, mglB, mglC, mglR, mgsA, mgtA, mhpA,mhpB, mhpC, mhpD, mhpE, mhpF, mhpR, miaA, miaD, micF, minC, minD, minE,mioC, mltA, mltB, mltC, mltD, mmrA(rhlB), mng, mntA, moaA, moaB, moaC,moaD, moaE, mobA, mobB, moc, modA, modB, modC, modE, modF, moeA, moeB,mog, molR, motA, motB, mpl, mppA, mprA, mraA, mraY, mrcA, mrcB, mrdA,mrdB, mreB, mreC, mreD, mrp, mrr, msbA, msbB, mscL, msrA, msyB, mtg,mtgA, mtlA, mtlD, mtlR, mtr, mttA, mttB, mttC, mukB, mukE, mukF, mul,murA, murB, murC, murD, murE, murF, murG, murH, murI, mutG(putative),mutH, mutL, mutM, mutS, mutT, mutY, nac, nadA, nadB, nadC, nadE, nagA,nagB, nagC, nagD, nagE, nalB, nalD, nanA, nanE, nanK, nanR, nanT, napA,napB, napC, napD, napF, napG, napH, narG, narH, narI, narJ, narK, narL,narP, narQ, narU, narV, narW, narX, narY, narZ, ndh, ndk, neaB, nei,nemA, nfi, nfnA, nfnB, nfo, nfrA, nfrB, nfrD, nfsA, nhaA, nhaB, nhaR,nikA, nikB, nikC, nikD, nikE, nirB, nirC, nirD, nlpA, nlpB, nlpC, nlpD,nmpC(qsr′), non, npr, nrdA, nrdB, nrdD, nrdE, nrdF, nrdG, nrfA, nrfB,nrfC, nrfD, nrfE, nrfF, nrfC, nth, ntpA, nuoA, nuoB, nuoC, nuoE, nuoF,nuoG, nuoH, nuoI, nuoJ, nuoK, nuoL, nuoM, nuoN, nupC, nupG, nusA, nusB,nusG, nuvA, nuvC, ogrK, ogt, ompA, ompC, ompF, ompG, ompR, ompT, ompX,oppA, oppB, oppC, oppD, oppE, oppF, opr, ops, oraA, ordL, orf-23(purB,reg)orf195(nikA-reg), orn, osmB, osmC, osmE, osmY, otsA, otsB, oxyR,oxyS, pabA, pabB, pabC, pac, pal, panB, panC, panD, panF, parC, parE,pat, pbpG, pck, pcm, pcnB, pdhR, pdxA, pdxB, pdxH, pdxJ, pdxK, pdxL,pdxY, pepA, pepD, pepE, pepN, pepP, pepQ, pepT, pfkA, pfkB, pflA, pflB,pflC, pflD, pfs, pgi, pgk, pgl, pgm, pgpA, pgpB, pgsA, pheA, pheP, pheS,pheT, pheU, pheV, phnC, phnD, phnE, phnF, phnG, phnH, phnI, phnJ, phnK,phnL, phnM, phnN, phnO, phnP, phoA, phoB, phoE, phoH, phoP, phoQ, phoR,phoU, phrB, phxB, pin, pioO, pit, pldA, pldB, plsB, plsC, plsX, pmbA,pncA, pncB, pnp, pntA, pntB, pnuC, poaR, polA, polB, popD, potA, potB,potC, potD, potE, potF, potG, potH, potI, poxA, poxB, ppa, ppc, pphA,pphB, ppiA, ppiB, ppiC, ppk, pppA, pps, ppx, pqiA, pqiB, pqqL, pqqM,prc, prfA, prfB, prfC, priA, priB, priC, priC, prlZ, prmA, prmB, proA,proB, proC, proK, proL, proM, prop, proQ, proS, proT, proV, proW, proX,prpA, prpC, prpR, prr, prs, psd, psiF, pspA, pspB, pspC, pspE, pspF,pssA, pssR, pstA, pstB, pstC, pstS, psu, pta, pth, ptrA, ptrB, ptsG,ptsH, ptsI, ptsN, ptsP, purA, purB, purC, purD, purE, purF, purH, purK,purL, purM, purN, purP, purR, purT, purU, pus, putA, putP, pykA, pykF,pyrB, pyrC, pyrD, pyrE, pyrF, pyrG, pyrH, pyrI, qmeC, qmeD, qmeE, qor,queA, racC, racR, radA, radC, ranA, rarD, ras, rbfA, rbn, rbsA, rbsB,rbsC, rbsD, rbsK, rbsR, rcsA, rcsB, rcsC, rcsF, rdgA, rdgB, recA, recB,recC, recD, recE, recF, recG, recJ, recN, recO, recQ, recR, recT, relA,relB, relE, relF, relX, rep, rer, rfaB, rfaC, rfaD, rfaF, rfaG, rfaH,rfaI, rfaJ, rfaK, rfaL, rfaP, rfaQ, rfaS, rfaY, rfaZ, rfbA, rfbB, rfbC,rfbD, rfbX, rfc, rfe, rffA, rffC, rffD, rffE, rffG, rffH, rffM, rffT,rhaA, rhaB, rhaD, rhaR, rhaS, rhaT, rhlB, rhlE, rho, ribA, ribB, ribC,ribD, ribE, ribF, ridA, ridB, rimB, rimC, rimD, rimE, rimG, rimH, rimI,rimJ, rimK, rimL, rimM, rit, rlpA, rlpB, rluA, rluC, rluD, rmf, rna,rnb, rnc, rnd, rne, rnhA, rnhB, rnk, rnpA, rnpB, rnr, rnt, rob, rorB,rpe, rph, rpiA, rpiB, rpiR, rplA, rplB, rplC, rplD, rplE, rplF, rplI,rplJ, rplK, rplL, rplM, rplN, rplO, rplP, rplQ, rplR, rplS, rplT, rplU,rplV, rplW, rplX, rplY, rpmA, rpmB, rpmC, rpmD, rpmE, rpmF, rpmG, rpmH,rpmI, rpmJ, rpoA, rpoB, rpoC, rpoD, rpoE, rpoH, rpoN, rpoS, rpoZ, rpsA,rpsB, rpsC, rpsD, rpsE, rpsF, rpsG, rpsH, rpsI, rpsJ, rpsK, rpsL, rpsM,rpsN, rpsO, rpsP, rpsQ, rpsR, rpsS, rpsT, rpsU, rrfA, rrfB, rrfC, rrfD,rrfE, rrfF, rrfG, rrfH, rrlA, rrlB, rrlC, rrlD, rrlE, rrlG, rrlH, rrmA,rrsA, rrsB, rrsC, rrsD, rrsE, rrsG, rrsH, rsd, rseA, rseB, rseC, rspA,rspB, rssA, rssB, rsuA, rtcA, rtcB, rtcR, rtn, rus(qsr′), ruvA, ruvB,ruvC, sad, sanA, sapA, sapB, sapC, sapD, sapF, sbaA, sbcB, sbcC, sbcD,sbmA, sbmC(gyrI), sbp, sdaA, sdaB, sdaC, sdhA, sdhB, sdhC, sdhD, sdiA,sds, secA, secB, secD, secE, secF, secG, secY, selA, selB, selC, selD,semA, segA, serA, serB, serC, serR serS, serT, serU, serV, serW, serX,sfa, sfcA, sfiC, sfsA, sfsB, shiA, sipC, sipD, sir, sixA, sloB, slp,slr, slt, slyD, slyX, smp, smtA, sodA, sodB, sodC, sohA, sohB, solA,soxR, soxS, speA, speB, speC, speD, speE, speF, speG, spf, spoT, sppA,spr, srlA, srlB, srlD, srlE, srlR, srmB, srnA, ssaE, ssaG, ssaH, ssb,sseA, sseB, sspA, sspB, ssrA, ssrS, ssyA, ssyD stfZ, stkA, stkB, stkC,stkD, stpA, strC, strM, stsA, sucA, sucB, sucC, sucD, sufi, sugE, suhA,suhB, sulA, supQ, surA, surE, syd, tabC, tag, talA, talB, tanA, tanB,tap, tar, tas, tauA, tauB, tauC, tauD, tbpA, tdcA, tdcB, tdcC, tdcD,tdcE, tdcF, tdcG, tdcR, tdh, tdi tdk, tehA, tehB, tesA, tesB, tgt, thdA,thdC, thdD, thiB?, thiC, thiD, thiE, thiF, thiG, thiH, thiI, thiJ, thiK,thiL, thiM, thrA, thrB, thrC, thrS, thrT, thrU, thrV, thrW, thyA, tig,tktA, tktB, tldD, tinA, tmk, tnaA, tnaB, tnaC, tnm, tol-orf1, tol-orf2,tolA, tolB, tolC, tolD, tolE, tolI, tolJ, tolM, toIQ, toIR, tonB, topA,topB, torA, torC, torD, torR, torS, torT, tpiA, tpr, tpx, treA, treB,treC, treF, treR, trg, trkA, trkD, trkG, trkH, trmA, trmB, trmC, trmD,trmE, trmF, trmH, trmU, trnA, trpA, trpB, trpC, trpD, trpE, trpR, trpS,trpT, truA, truB, trxA, trxB, trxC, tsaA, tsf, tsmA, tsr, tsx, ttdA,ttdB, ttk, tufA, tuffB, tus, tynA, tyrA, tyrB, tyrP, tyrR, tyrS, tyrT,tyrU, tyrV, ubiA, ubiB, ubiC, ubiD, ubiE, ubiF, ubiG, ubiH, ubiX, ucpA,udk, udp, ugpA, ugpB, ugpC, ugpE, ugpQ, uhpA, uhpB, uhpC, uhpT, uidA,uidB, uidR, umuC, umuD, ung, upp, uppS, ups, uraA, usg-1, usbA, uspA,uup, uvh, uvrA, uvrB, uvrC, uvrD, uvs, uxaA, uxaB, uxaC, uxuA, uxuB,uxuR, valS, valT, valU, valV, valW, valX, valY, valZ, vsr, wrbA, xapA,xapB, xapR, xasA, xerC, xerD, xni, xseA, xseB, xthA, xylA, xylB, xylE,xylF, xylG, xylH, xylR, yccA, yhhP, yihG, yjaB, f147, yjaD, yohF, yqiE,yrfE, zipA, zntA, znuA, znuB, znuC, zur, and zwf.

Non-limiting examples of mouse genes include: Ilr1, Ilr2, Gas10, Tnp1,Inhbb, Inha, Creb1, Mpmv34, Acrd, Acrg, Il110, Otf1, Rab11b-r, Ab11,ald, Amh-rs1, Bcl2B, Cchlla3, Ccnb1-rs2, Gpcr16, Htr5b, Idd5, Igfbp2,Igfbp5, I18rb, Kras2-rs1, Mov7, Mpmv6, Mpmv16, Mpmv22, Mpmv25, Mpmv29,Mpmv42, Mtv7, Mtv27, Mtv39, Oprk1, Otf3-rs1, Otf8, Otf11-rs1, Ptgs2,Ren1, Ren2, Ril3, Sxv, Taz4-rs1, Tgfb2, Wnt6, Xmmv6, Xmmv9, Xmmv36,Xmmv61, Xmmv74, Xmv21, Xmv32, Xmv41, I12ra, Ab1, Mpmv3, Rap1a-ps2, anx,Mpmv43, Ryr3, Ras12-4, Adra2b, Avp, Glvr1, Il1a, Il1b, Mpmv28, Oxt,Pcsk2, a, Xmv10, Tcf4, Acra, Acra4, Ak1, Bdnf, bs, Cyct, Cyp24, Dbh,Fshb, Gcg, Gdf5, Gnas, Gpcr8, Grin1, Hcs4, Hior2, Hsp84-2, Idd12, Ilrn,Jund2, Kras3, Mc3r, Mpmv14, Mtv40, Mxi1-rs1, Otf3-rs2, Ptgs1, Ptpra,Rapsn, Src, Svp1, Svp3, Tcf3b, Wt1, Xmmv71, Xmv48, Ccna, Fgf2, Fth-rs1,Csfm, Mov10, Egf, Acrb2, Cap1, Crh, Fim3, Fps11, Glut2, Gper2, Gria2,Hsd3b-1, Hsd3b-2, Hsd3b-3, Hsd3b-4, Hsp86-ps2, Idd3, I12, I17, Mpvmv9,Mpmv20, Mtv4.8, Ngfb, Npra, Nras, Nras, Ntrk, Otf3-rs3, Otf3-rs4, Rap1a,Tshb, Xmmv22, Xmmv65, Mos, Ras12-7, Lyr, Ifa, Ifb, Jun, azh, db, Ipp,Mp1, Do1, Ak2, Ccnb1-rs4, Cdc211, Cga, Fgr, Foc1, Fps12, Gabrr1, Gabrr2,Gdf6, Glut1, Gnb1, Gpcr14, Grb2-ps, Grik3, Grik5, Hsp86-1ps4, Htr1da,Htr1db, Idd9, Ifa1, Ifa2, Ifa3, Ifa4, Ifa5, Ifa6, Ifa7, Ifa8, Ifa9,Ifa10, Lap18, Lmyc1, Mpmv19, Mpmv44, Mtv13, Mtv14, Mtv17, Nppb, Otf6,Otf7, Ri12, Ski, Tnfr2, Wnt4, Xmmv8, Xmmv23, Xmmv62, Xmv1, Xmv2, Xmv8,Xmv9, Xmv14, Xmv44, Xpa, Tec, Fgf5, Nos1, Tcf1, Epo, Gnb2, Flt1, Flt3,Ache, Adra2c, Adrbk2, Afp, Alb1, Ccnb1-rs1, Clock, Cyp3, Cyp3a11,Cyp3a13, Drd1b, Drd5, Fgfr3, Flk1, Gc, Gnrhr, Gpcr1, Hcs5, Hnf1, Htr5a,I15r, I16, Kit, Ltrm3, Mgsa, Mpmv7, Mpmv13, Mpmv23, Mtv32, Mtv41, Pdgfa,Pdgfra, Por, Txk, Xmmv3, Xmmv5, Xmmv52, Xmv17, Xmv28, Xmv34, Xmv38,Xmv45, Zp3, Trh, Raf1, Fth-rs2, Ntf3, Kras2, Pthlh, Mov1, Alox5, Braf2,Cftr, Egr4, Fps110, Fgf6, Gdf3, Ghrfr, Glut3, Grin2a, Hior3, Hoxa10,hop, Ica1, I15r, Int41, Itpr1, Krag, Mad, Met, Mi, Mtv8, Mtv23, Mtv29,Mtv33, Mtv34, Nkna, Npy, ob, Otf3-rs5, Tgfa, Tnfr1, Wnt2, Wnt5B, Wnt7A,Xmmv27, Xmv24, Xmv61, Fosb, Ryr1, Ngfa, Ufo, Xrcc1, Abpa, Abpga, Gabra4,Gas2, Acra7, Ccnb1-rs7, Egfbp3, Xmv30, Zp2, Fes, Pcsk3, Calc,Ccnb1-rs10, Pth, Ad, Bc13, Cea, Cea2, Cea3, Cea4, Cea5, Cea6, Cebp, Dm9,Dm15, Drd4, Egfbp1, Egfbp2, Ercc2, Fgf3, Fgfr2, Gabra5, Gabrb3, Gtx,Hcs1, Igf1r, Igf2, I14r, Ins2, Int40, Lhb, Mpmv1, Mtv1, Mtv35, Ngfg,Ntf5, Otf2, 2, Pkcc, Ras14, Rras, Ryr, Svp2, Tcf3g, Tgfb1, tub, Xmmv31,Xmmv35, Xmmv73, Xmv33, Xmv53, Taz83, Adrb3, Junb, Jund1, Me1,Gpcr19-rs2, Agt, Cadp, Ccnb1-rs9, E, Fgfr1, Gas6, Gnb-rs1, Hcs2, Insr,Maf, Mov34, Mpmv21, Mpmv41, Mtv21, Mtnr1a, Plat, Ras15-2, Ras16, Sntb2,Xmmv29, Xmv12, Xmv26, Xmv62, Epor, Gpcr13, Otf11, Pthr, Acra3, Acra5,Acrb4, Camk1, Cdc25Mm, Crbp, Crbp2, Csk, Cyp11a, Cyp19, Drd2, Ets1l,Fli1, Gnai2, Gnat1, Gpcr6, Gria4, Hgf1, Hior1, Hpx, Hsp86-1ps3, Hst2,Idd2, I11bc, Lag-rs1, Lap18-rs1, M11, Mpmv27, Penk, Pgr, Ras12-2, Tp11,Trf, Xmmv2, Xmmv67, Xmv15, Xmv16, Xmv25, Xmv60, Mgf, Amh, Braf, Cdc2a,Dmd1, Estr, Fps13, Fps14, Fps15, Gli, Gpcr17, Grik2, Ifgr, Igf1, Mpmv5,Mpmv12, Mpmv40, Myb, Oprm, Pg, Pmch, Ros1, Xmv31, Xmv51, Xmv54, Camk2b,Egfr, Int6, Lif, Mtv44, Ews, Csfgm, Flt4, I13, I14, I15, Irf1, Gria1,Glut4, Crhr, Csfg, Mov9, Xmv20, Acrb, Mpmv4, Mpmv15, Ngfr, Nos2, Rara,Taz4, Tcf2, Xmv42, Mtv3, Adra1, Crko, df, Erbb2, Gabra1, Gabra6, Gabrg2,Gh, Glra1, Grb2, Hnf1b, Hsp86-ps1, Idd4, Igfbp1, Igfbp3, I113, Int4,Mpmv2, Mpmv8, Mpmv18, Mtv45, nu, Pkca, Rab1, Re1, Shbg, Tcf7, Thra,Tnz1, Trp53, Wnt3, Wnt3A, Xmv4, Xmv5, Xmv47, Xmv49, Xmv63, Akt, Amh-rs4,Ccs1, Fps16, Fos, Gdf7, Hcs3, Hsp70-2, Hsp84-3, Hsp86-1, hyt, Ltrm1,Max, Mpmv11, Mpmv24, Mtv9, Mtv30, Pomc1, Tcf3a, Tda2, Tgfb3, Tpo, Tshr,Xmmv21, Xmmv25, Xmmv34, Xmmv50, Gli3, Xmv55, Ryr2, Inhba, Gas1, Pcsk1,Amh-rs2, Ccnb1-rs6, Ccnb1-rs13, Crhpb, Dat1, Drd1a, Fgfr4, Fps17, Fim1,Gpcr15, Gpcr18, Hbvi, Hilda, Htr1a, Idd1, I19, Ltrm4, Mak, mes, P11,P12, Pr1, Ra1, Rasa, Srd5a1, Tpbp, Xmv13, Xmv27, Rarb, Rbp3, Htr2, Rb1,Acra1, Camkg, Cch11a2, Ccnb1-rs5, Ccnb1-rs12, Gnrh, Mtv11, Nras-ps,Otf3-rs6, Plau, Ptprg, Trp53-ps, Wnt5A, Xmv19, Ghr, I17r, Lifr, Mlvi2,Prlr, Myc, Ril1, cog, Amh-rs7, I12rb, Pdgfb, Acr, CP2, Rarg, Sp1-1,Wnt1, Afr1, Atf4, Bzrp, Ccnb1-rs11, Cyp11b, I13rb1, I13rb2, Ins3, Itga,Mlvi1, Mlvi3, Mtv36, Pdgfec, Svp5, Tef, Trhr, Wnt7B, Xmmv55, Xmmv72,Xmv37, Tnp2, Ets2, Casr, Chuck-rs1, din, Drd3, Erg, G22p1, Gap43, Gas4,Grik1, Htr1f, Ifgt, Int53, Ltrm2, Mpmv17, Mtv6, Mtvr1, Pit1, Xmv3,Xmv35, Xmv50, Igf2r, Mas, Tcd3, Glp1r, Idd1, Tla, Aeg1, Ccnb1-rs3,Cdc2b, Csi, Cyp21, Cyp21-ps1, Fps18, Gna-rs1, Gpcr19-rs1, Grr1, Grr2,Hom1, Hsc70t, Hsp70, Hsp70-1, Hsp70-3, Hsp84-1, Hst1, Hst4, Hst5, Hst6,Hye, Int3, Itpr3, Lap18-rs2, Otf3, Ptprs, Rab11b, Ras12-1, Ras12-3,Ras13, Rrs, Rxrb, Tas, Tcd1, Tcd2, Tera1, Tla-rs, Tnfa, Tnfb, Tpx1,Tpx2, Xmmv15, Xmv36, Xmv57, Csfmr, Pdgfrb, Adrb2, Apc, Camk2a, Camk4,Dcc, Fgf1, Gna1, Gpcr7, Gr11, Grp, Hsp74, Mcc, Mtv2, Mtv38, Ptpn2, Tp12,Xmv22, Xmv23, Xmv29, Fth, Csfgmra, Mxi1, Adra2a, Adrb1, Adrbk1, Chuck,Cyp17, Gna14, Gnb-ps1, Hcs6, Htr7, Ide, Ins1, Lpc1, Pomc2, Seao, T1x1,Xmmv42, Xmv18, Tcfe3, Araf, Avpr2, mdx, Ar, Zfx, Otf9, Ccg1, Ccnb1-rs8,Fps19, Gabra3, Glra2, Glra4, Gria3, Grpr, Hsp74-ps1, Hst3, Htr1c, I12rg,Mov14, Mov15, Mtv28, Otf3-rs8, Sts, Sxa, Sxr, Xta, Tdy, Hya, Zfy1, Zfy2,Mov15, Mov24, Mtv31, Mtv42, Sdma, Spy, Sts, Sxa, Sxr, XmmvY, Xmv7,Xmv11, and Xmv40.

Non-limiting examples of Phaseolus vulgaris genes include: Acc, ace,Adk, Am, Amv-1, Amv-2, Ane, aph, Arc, Are, arg, Ar1 (Arc), asp, B, bc-u,bc-1.sup.1, bc-1.sup.2, bc-2.sup.1, bc-2.sup.2, be-3, Bcm, Beg, Bip,blu, Bpm, Bsm, By-1, By-2, C, C/c, c.sup.cr, C.sup.cir, C.sup.ma (M,R.sup.ma), C.sup.r, C.sup.res, C.sup.rho, C.sup.st, [C.sup.st R Acc](Aeq), c.sup.0 (inh, i.sub.e), [c.sup.0 Prp.sup.i] (Pm, c.sup.ui, Nud),[c.sup.uprp.sup.st] (prp.sup.st), [C Prp] (Prp), c.sup.v, [C R] (R), [Cr] (r), Ca, Cam, Cav, cc, ch1, c1, cm1, Co-1 (A), Co-2 (Are), Co-3(Mexique 1), Co-3.sup.2, Co-4 (Mexique 2), Co-5 (Mexique 3), Co-6, Co-7,cr-1 cr-2, cry, cs, Ct, ctv-1 ctv-2, cyv (by-3), D (Can, Ins), Da, Db,def, dgs (g1, 1e), dia, Diap-1, Diap-2, diff, dis, D1-1 D1-2 (DL.sub. 1DL.sub.2), do, ds (te), dt-1.sup.a dt-2.sup.a, dt-1.sup.b dt-2.sup.b,dw-1 dw-2, Ea Eb, ers (restr), ers-2, Est-1, Est-2, exp, F, Fa, fast, FbFc, fa fb fc, Fcr, Fcr-2, fd, Fe-1 Fe-2, Fin (in), Fop-1, Fop-2, Fr,Fr-2, G (Flav, Ca, Och), Ga, gas, glb, Gpi-c1, Gr, Hb1 (L.sub.HB-1),Hbnc (SC.sub.HB-1), Hbp (PD.sub.HB-1), hmb, Hss, Hsw, Ht-1 Ht-2 (L-1L-2), I, Ia Ib, ian-1 ian-2 (ia), lbd, ico, Igr (Ih), ilo, ip, iter, iv,iw, J (Sh), Ke, L, la, Lan, Ld, Lds (Ds), Lec, Li (L), lo, Ir-1 lr-2,mar, Me, Mel (Me), Mel-2 (Me-2), mel-3 (me-3), Mf, mi, mia, Mic (Mip),miv, Mrf, Mrf.sup.2, mrf, ms-1, Mue, mu mutator, Nag, Nd-1 Nd-2 (D-1D-2), nie, and (sym-1), and-2, No, nts (nod), Nudus, ol, P, p.sup.gri(Gri, v.sup.Pal), pa, pc, pg (pa.sub.1), Pha, Pmv, ppd (neu), Pr, prc(pc), Prx, punc, ram, Rbcs (rbcS), rf-1, rf-2, rf-3, rfi (i), Rfs (m),Rk, rk, rk.sup.d (lin), rn-1 rn-2 (r r), md, Ro, Sal, sb, sb.sup.ms,sb-2, sb-3, si1, Skdh, s1, Smv, St, Sur, sw-1 sw-2, T, t (z-1), Th-1Th-2, Tm, To, Tor (T), Tr, tri, trv, Ts, tw, uni, Uni-2, uni.sup.nde,uni.sup.nie, Ur-1, Ur-2, Ur-2.sup.2, Ur-3 (Ur-3, Ur-4), Ur-3.sup.2,Ur-4, (Up-2, Ur-C), Ur-5, (B-190), Ur-6 (Ur.sub.a, Ur-G), Ur-7(R.sub.B11), Ur-8 (Up-1), Ur-9 (Ur.sub.p), us, V (B1), v.sup.lae (Cor),v, var, vi (vir.sub.f), wb, Wmv, X.sup.su, y, and Z.

Non-limiting examples of Saccharomyces cerevisiae genes include: PRE3,PUP1, PUP3, PRE2, PRE10, PRE1, PRE8, SCL1, PUP2, PRE5, PRE7, PRE4, RPT2,RPT3, RPN3, RPN11, RPN12, RPT6, RPN1, RPN2, RPT1, RPT5, RPT4, SKI6,RRP4, DIS3, TSC10, RAT1, GND1, EXO70, ERG10, ACC1, RPP0, ACT1, ARP100,ARP3, PAN1, ARP2, ARP4, ARP9, SPE2, CYR1, ALA1, TPS1, TUB1, ABF1, DED81,NIP1, YHC1, SNU71, ATM1, MAK5, ROK1, DED1, SPB4, AUR1, PSE1, ALG1, TUB2,BPL1, MSL5, ERG24, ERG26, ERG25, CMD1, HCA4, SHE9, SHE10, CAK1, PIS1,CHO1, CDS1, ESR1, NUD1, CDC47, CDC13, CDC37, CDC1, CDC4, CDC20, CDC6,CDC46, CDC3, KAR1, BBP1, HRP1, CCT2, CCT3, HSP10, SMC1, SMC2, CHC1,CFT2, CLP1, COP1, SEC26, SEC27, RET2, SEC21, COF1, CCT4, CCT1, CCT6,SEC24, SECT, PCF11, RNA15, RNA14, FIP1, YSH1, TFB4, TSM1, APC2, APC5,SEC31, TAF47, TAP42, MPP10, CDC53, CKS1, CDC28, KIN28, CNS1, ERG11,DBP10, DBP8, PRO3, DYS1, ALR1, TID3, DNA2, SSL2, RAD3, RFA3, RFA2, RFA1,RFC4, RFC5, RFC3, RFC2, RFC1, TOP2, RAP1, RPC25, PRI2, PRI1, POL1,POL12, HUS2, CDC2, POL2, DPB2, RPB10, RPA135, RPA190, RPA43, RPB8,RPO26, RPB5, RPC40, RPC19, SRB7, SRB4, RGR1, RPB11, SRB6, RPB2, RPB7,RPO21, RET1, RPO31, RPC31, RPC34, RPC53, RPC82, RPB12, RPB3, DPM1, DIP2,RNT1, CDC8, CDC14, DUT1, UBA2, UBA1, UBC9, CDC34, ENP1, ERD2, SSS1,SEC61, SEC63, SEC62, GNA1, GPI8, DAM1, DUO1, IRR1, PRP3, TIM9, HSH49,SUP35, EXM2, MEX67, ERG9, ERG20, FAS2, FAS1, NOP1, FAD1, AOS1, FBA1,NCB2, BRN1, TUB4, GDI1, GOG5, SRM1, CDC25, SPT16, YIF2, BET4, CDC43,MRS6, BET2, PRO1, GLN1, GLN4, GRS1, YIP1, FOL2, GPA1, CDC42, SAR1, YPT1,SEC4, GSP1, TEM1, RHO1, CDC24, RNA1, GUK1, VMA16, PMA1, HKR1, SIS1,MGE1, HSP60, HSF1, HAS1, MOT3, HTS1, ESA1, HSL7, HOM6, RIB7, SLY1, CSL4,PUR5, CSE1, IPP1, MDM1, USO1, SOF1, MAK11, LAS1, TEL2, DPB11, SGD1,FAL1, MTR3, MTR4, SPP2, SIK1, RRP7, POP4, RRP1, POP3, BFR2, CDC5, NRD1,MET30, MCM6, RRP46, SAS10, SCC2, ECO1, PRP43, BET3, BET5, STN1, NFS1,IDI1, SRP1, KAP95, CBF2, SKP1, CEP3, CTF13, ERG7, KRS1, PSA1, PMI40,ALG2, SSF1, MED7, RSC4, CDC54, MCM2, AFG2, ERG12, MVD1, CDC48, MHP1,ERV1, SSC1, TIM44, TIM17, TIM23, TOM22, TOM40, MAS1, MCD1, MMC1, STU1,JAC1, ABD1, CEG1, PAB1, MTR2, SEC16, ROT1, INO1, MLC1, MYO2, GPI2,SPT14, NAT2, NMT1, TRM1, NCP1, NBP1, ACF2, SPP41, NUT2, LCP5, PRP19,NMD3, RFT1, NNF1, NDC1, CRM1, KAR2, NIP29, NAB2, NIC96, NUP145, NUP49,NUP57, NUP159, NSP1, NUP82, CDC39, NPL4, POP7, NTF2, MAK16, NPL3, NOP2,NOP4, NHP2, NOP10, GAR1, NBP35, WBP1, STT3, SWP1, OST2, OST1, ORC1,ORC6, ORC5, ORC4, ORC3, RRR1, SAT2, PWP2, PEX3, TOR2, PIK1, SEC14, STT4,MSS4, PCM1, GPM1, SEC53, ERG8, YPD1, PAP1, NAB3, RRN7, SEN1, CFT1,PRP11, PRP21, PRP39, PRP24, PRP9, SLU7, PRP28, PRP31, IFH1, PTA1, SUB2,FM11, MAS2, ESS1, PFY1, POL30, POP1, PDI1, RAM2, CDC7, SMP3, CDC15,YTH1, QRI2, YAE1, SFI1, SEC1, BET1, SEC6, SEC13, SEC2, SEC8, CBF5,CDC19, YRB1, RHC18, DBF4, SDS22, MCM3, CEF1, ALG11, GAA1, MOB1, NIP7,TIP20, SEC5, SEC10, GPI10, RRP3, CDC45, DIB1, MIF2, HOP2, PBN1, NOP5,RPP1, POP5, POP8, POP6, ERO1, MPT1, DNA43, ESP1, SMC3, LST8, STS1, RPM2,RNR1, RNR2, RNR4, RPS20, RPL25, RPL3, RPL30, RPL32, RPL37A, RPL43A,RPL5, RPL10, RPS3, CET1, YRA1, SNM1, GLE1, DBP5, DRS1, DBP6, BRR2, RRN3,RRN6, RRN11, MED6, PRP16, RPR2, DIM1, RRP43, RRP42, RRP45, SEC20, BOS1,CDC12, GLC7, PKC1, IPL1, SGV1, NRK1, RAD53, LCB2, LCB1, MPS1, SES1,SPC3, SEC11, RIO1, ARP7, NEO1, YJU2, POB3, ARH1, IQG1, HRT1, HYM1,MAK21, FUN20, FUN9, NBN1, STB5, YIF1, SMX4, YKT6, SFT1, SMD1, PRP6,LSM2, NUF1, SPC97, SPC42, SPC98, CDC31, SPC19, SPC25, SPC34, SPC24,NUF2, PRP40, MCD4, ERG1, SMC4, CSE4, KRR1, SME1, TRA1, RLP7, SCH9, SMD3,SNP2, SSF2, SPC72, CDC27, CDC23, CDC16, APC1, APC11, APC4, ARC19, RPN6,RPN5, RSC6, RSC8, STH1, SFH1, TIM12, TIM22, TIM10, SQT1, SLS1, JSN1,STU2, SCD5, SSU72, ASM4, SED5, UFE1, SYF1, SYF2, CCT5, TBF1, TOA2, TOA1,SUA7, TAF90, TAF61, TAF25, TAF60, TAF17, TAF145, TAF19, TAF40, TAF67,TFA2, TFA1, FCP1, TFG1, TFG2, TFB1, CCL1, SSL1, TFB3, TFB2, PZF1, BRF1,TFC5, TFC4, TFC3, TFC7, TFC6, TFC1, SPT15, THI80, THS1, SPT6, SPT5,ROX3, REB1, MCM1, MED4, MOT1, MED8, EFB1, YEF3, SUI1, CDC95, TIF11,SUI3, GCD11, SUI2, GCD6, GCD7, GCD2, GCD1, RPG1, GCD10, PRT1, TIF34,CDC33, TIF5, SUP45, GCD14, TIM54, SEC17, TPT1, TRL1, CCA1, SEN54, SEN2,SEN15, SEN34, WRS1, SLN1, TYS1, SNU56, PRP42, CUS1, PRP4, PRP8, SNU114,USS1, UFD1, SMT3, RSP5, QRI1, ALG7, UGP1, VTI1, VAS1, SEC18, CTR86, andZPR1.

2. Viruses

The microorganisms provided herein include viruses. Such virusestypically have one or more of the microorganism characteristics providedherein. For example, viruses provided herein can have attenuatedpathogenicity, reduced toxicity, preferential accumulation inimmunoprivileged cells and tissues, such as tumor, ability to activatean immune response against tumor cells, immunogenic, replicationcompetent, and are able to express exogenous proteins, and combinationsthereof. In some embodiments, the viruses have an ability to activate animmune response against tumor cells without aggressively killing thetumor cells.

The viruses provided herein can be cytoplasmic viruses, such aspoxviruses, or can be nuclear viruses such as adenoviruses. The virusesprovided herein can have as part of their life cycle lysis of the hostcell's plasma membrane. Alternatively, the viruses provided herein canhave as part of their life cycle exit of the host cell by non-lyticpathways such as budding or exocytosis. The viruses provided herein cancause a host organism to develop an immune response to virus-infectedtumor cells as a result of lysis or apoptosis induced as part of theviral life cycle. The viruses provided herein also can be geneticallyengineered to cause a host organism to develop an immune response tovirus-infected tumor cells as a result of lysis or apoptosis, regardlessof whether or not lysis or apoptosis is induced as part of the virallife cycle. In some embodiments, the viruses provided herein can causethe host organism to mount an immune response against tumor cellswithout lysing or causing cell death of the tumor cells.

One skilled in the art can select from any of a variety of viruses,according to a variety of factors, including, but not limited to, theintended use of the virus (e.g., exogenous protein production, antibodyproduction or tumor therapy), the host organism, and the type of tumor.

a. Cytoplasmic Viruses

The viruses provided herein can be cytoplasmic viruses, where the lifecycle of the virus does not require entry of viral nucleic acidmolecules in to the nucleus of the host cell. A variety of cytoplasmicviruses are known, including, but not limited to, pox viruses, Africanswine flu family viruses, and various RNA viruses such as picornaviruses, calici viruses, toga viruses, corona viruses and rhabdoviruses. In some embodiments, viral nucleic acid molecules do not enterthe host cell nucleus throughout the viral life cycle. In otherembodiments, the viral life cycle can be performed without use of hostcell nuclear proteins. In other embodiments, the virulence orpathogenicity of the virus can be modulated by modulating the activityof one or more viral proteins involved in viral replication.

i. Poxviruses

In one embodiment, the virus provided herein is selected from the poxvirus family. Pox viruses include Chordopoxyirinae such asorthopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus, Leporipoxvirus,suipoxvirus, molluscipoxvirus and yatapoxvirus, as well asEntomopoxyirinae such as entomopoxvirus A, entomopoxvirus B, andentomopoxvirus A. Chordopoxyirinae are vertebrate poxviruses and havesimilar antigenicities, morphologies and host ranges; thus, any of avariety of such poxviruses can be used herein. One skilled in the artcan select a particular genera or individual chordopoxyirinae accordingto the known properties of the genera or individual virus, and accordingto the selected characteristics of the virus (e.g., pathogenicity,ability to elicit and immune response, preferential tumor localization),the intended use of the virus, the tumor type and the host organism.Exemplary chordopoxyirinae genera are orthopoxvirus and avipoxvirus.

Avipoxviruses are known to infect a variety of different birds and havebeen administered to humans. Exemplary avipoxviruses include canarypox,fowlpox, juncopox, mynahpox, pigeonpox, psittacinepox, quailpox,peacockpox, penguinpox, sparrowpox, starlingpox, and turkeypox viruses.

Orthopoxviruses are known to infect a variety of different mammalsincluding rodents, domesticated animals, primates and humans. Severalorthopoxviruses have a broad host range, while others have narrower hostrange. Exemplary orthopoxviruses include buffalopox, camelpox, cowpox,ectromelia, monkeypox, raccoon pox, skunk pox, tatera pox, uasin gishu,vaccinia, variola and volepox viruses. In some embodiments, theorthopoxvirus selected can be an orthopoxvirus known to infect humans,such as cowpox, monkeypox, vaccinia or variola virus. Optionally, theorthopoxvirus known to infect humans can be selected from the group oforthopoxviruses with a broad host range, such as cowpox, monkeypox, orvaccinia virus.

a. Vaccinia Virus

One exemplary orthopoxvirus is vaccinia virus. A variety of vacciniavirus strains are available, including Western Reserve (WR), Copenhagen,Tashkent, Tian Tan, Lister, Wyeth, IHD-J, and IHD-W, Brighton, Ankara,MVA, Dairen I, L-IPV, LC16M8, LC16MO, LIVP,WR 65-16, Connaught, New YorkCity Board of Health. Exemplary vaccinia viruses are Lister or LIVPvaccinia viruses. Any known vaccinia virus, or modifications thereofthat correspond to those provided herein or known to those of skill inthe art to reduce toxicity of a vaccinia virus. Generally, however, themutation will be a multiple mutant and the virus will be furtherselected to reduce toxicity.

The linear dsDNA viral genome of vaccinia virus is approximately 200 kbin size, encoding a total of approximately 200 potential genes. Viralgene expression can be divided into three stages. In the early stage,gene expression is mainly for viral replication, and for defense againstthe host's immune system. In the intermediate stage, genes not availablefor expression in the early stage can be expressed, including late stagetransactivators. In the late stage, active transcription is mainly forviral structural components for building mature viruses.

Vaccinia virus possesses a variety of features for use in cancer genetherapy and vaccination. It has a broad host and cell type range.Vaccinia is a cytoplasmic virus, thus, it does not insert its genomeinto the host genome during its life cycle. Unlike many other virusesthat require the host's transcription machinery, vaccinia virus cansupport its own gene expression in the host cell cytoplasm using enzymesencoded in the viral genome. The vaccinia virus genome has a largecarrying capacity for foreign genes, where up to 25 kb of exogenous DNAfragments (approximately 12% of the vaccinia genome size) can beinserted. The genomes of several of the vaccinia strains have beencompletely sequenced, and many essential and nonessential genesidentified. Due to high sequence homology among different strains,genomic information from one vaccinia strain can be used for designingand generating modified viruses in other strains. Finally, thetechniques for production of modified vaccinia strains by geneticengineering are well established (Moss, Curr. Opin. Genet. Dev. 3(1993), 86-90; Broder and Earl, Mol. Biotechnol. 13 (1999), 223-245;Timiryasova et al., Biotechniques 31 (2001), 534-540).

Historically, vaccinia virus was used to immunize against smallpoxinfection. More recently, modified vaccinia viruses are being developedas vaccines to combat a variety of diseases. Attenuated vaccinia viruscan trigger a cell-mediated immune response. Strategies such asprime/boost vaccination, vaccination with nonreplicating vaccinia virusor a combination of these strategies, have shown promising results forthe development of safe and effective vaccination protocols. Mutantvaccinia viruses from previous studies exhibit a variety ofshortcomings, including a lack of efficient delivery of the viralvehicle to the desired tissue only (e.g., specific accumulation in atumors), a lack of safety because of possible serious complications(e.g., in young children, eczema vaccinatum and encephalitis, and inadults disseminated or progressive vaccinia may result if the individualis severely immunodeficient).

b. Modified Vaccinia Viruses

Provided herein are vaccinia viruses with insertions, mutations ordeletions, as described more generally elsewhere herein. The vacciniaviruses are modified or selected to have low toxicity and to accumulatein the target tissue. Exemplary of such viruses are those from the LIVPstrain.

Exemplary insertions, mutations or deletions are those that result in anattenuated vaccinia virus relative to the wild type strain. For example,vaccinia virus insertions, mutations or deletions can decreasepathogenicity of the vaccinia virus, for example, by reducing thetoxicity, reducing the infectivity, reducing the ability to replicate,or reducing the number of non-tumor organs or tissues to which thevaccinia virus can accumulate. Other exemplary insertions, mutations ordeletions include, but are not limited to, those that increaseantigenicity of the microorganism, those that permit detection orimaging, those that increase toxicity of the microorganism (optionally,controlled by an inducible promoter). For example, modifications can bemade in genes that are involved in nucleotide metabolism, hostinteractions and virus formation. Any of a variety of insertions,mutations or deletions of the vaccinia virus known in the art can beused herein, including insertions, mutations or deletions of: thethymidine kinase (TK) gene, the hemagglutinin (HA) gene, the VGF gene(as taught in U.S. Pat. Pub. No. 20030031681); a hemorrhagic region oran A type inclusion body region (as taught in U.S. Pat. No. 6,596,279);Hind III F, F13L, or Hind III M (as taught in U.S. Pat. No. 6,548,068);A33R, A34R, A36R or B5R genes (see, e.g., Katz et al., J. Virology77:12266-12275 (2003)); SalF7L (see, e.g., Moore et cd., EMBO J. 199211:1973-1980); NIL (see, e.g., Kotwal et al., Virology 1989171:579-587); M1 lambda (see, e.g., Child et al., Virology. 1990174:625-629); HR, HindIII-MK, HindIII-MKF, HindIII-CNM, RR, or BamF(see, e.g., Lee et al., J. Virol. 1992 66:2617-2630); or C21L (see,e.g., Isaacs et al., Proc Natl Acad Sci USA. 1992 89:628-632).

c. The F3 Gene

In addition to the mutations known in the art, the vaccinia virusesprovided herein can have an insertion, mutation or deletion of the F3gene (SEQ ID No: 1; an exemplary F3 gene is provided in GenBankAccession No. M57977, which contains the nucleotide and predicted aminoacid sequences for LIVP strain F3; see also Mikryukov et al.,Biotekhnologiya 4:442-449 (1988)). For example, the F3 gene has beenmodified at the unique single NotI restriction site located within theF3 gene at position 35 or at position 1475 inside of the HindIII-Ffragment of vaccinia virus DNA strain LIVP (Mikryukov et al.,Biotekhnologiya 4 (1988), 442-449) by insertion of a foreign DNAsequence into the NotI digested virus DNA. As provided herein, aninsertion of a nucleic acid molecule, such as one containing lacZ, intothe NotI site of the F3 gene of the LIVP strain (nucleotides 1473-1480in M57977, or nucleotides 33-40 of SEQ ID NO: 1) can result in decreasedaccumulation of vaccinia viruses in non-tumorous organs of nude mice,including brain and heart, relative to wild type vaccinia virus. Thusfor use in the methods provided herein, vaccinia viruses can contain aninsertion, mutation or deletion of the F3 gene or a mutation of acorresponding locus. For example, as provided herein, F3-interruptedmodified LIVP vaccinia virus can selectively replicate in tumor cells invivo. Therefore, modified vaccinia viruses (e.g., modified strain LIVP)with the interrupted F3 gene can be used in the methods provided herein,such as methods of tumor-directed gene therapy and for detection oftumors and metastases.

Thus, provided herein are vaccinia viruses having a modification of theF3 gene. For example, the vaccinia viruses provided herein can containan insertion of foreign DNA into the F3 gene. An exemplary insertion offoreign DNA is an insertion at a site equivalent to the NotI site of theF3 gene in vaccinia strain LIVP, or at position 35 of SEQ ID NO:1. AnF3-modified vaccinia virus provided herein can colonize in tumorsspecifically, and therefore, can be used for tumor-specific therapeuticgene delivery. A GenBank data analysis with BLAST (Basic Local AlignmentSearch Tool) on nucleotide sequences of different strains of vacciniavirus was performed. Based on this analysis, it was found that invaccinia virus strain Copenhagen (Goebel et al., Virology 179 (1990),247-266) the NotI restriction site is located between two open readingframes (ORF) encoding F14L and F15L genes. Therefore, insertion offoreign genes into NotI site of the VV genome strain Copenhagen will notinterrupt any vital genes. In VV strain LIVP, the NotI restriction siteis located in the ORF encoding the F3 gene with unknown function(Mikryukov et Biotekhnologiya 4 (1988), 442-449). Thus, the insertion offoreign genes into the NotI site of the F3 gene interrupted the F3 gene.The ability to modify the F3 gene suggests that it may have anonessential role for virus replication. Although the F3 gene is likelynonessential for virus replication, the results of the animalexperiments suggest that interruption of the F3 gene is correlated withdecreased viral virulence, the inability to replicate in brain or ovary,and the ability to replicate preferentially in tumor tissue.

The F3 gene is conserved in a variety of different vaccinia virusstrains, including WR (nucleotides 42238-42387 of GenBank Accession No.AY243312.1, Ankara (nucleotides 37155-37304 of GenBank Accession No.U94848.1), Tian Tan (nucleotides 41808-41954 of GenBank Accession No.AF095689), Acambis 3000 (nucleotides 31365-31514 of GenBank AccessionNo. AY603355.1) and Copenhagen (nucleotides 45368-45517 of GenBankAccession No. M35027.1) strains. The F3 gene also is conserved in thelarger family of poxviruses, particularly among orthopoxviruses such ascowpox (nucleotides 58498-58647 of GenBank Accession No. X94355.2),rabbitpox (nucleotides 46969-47118 of GenBank Accession No. AY484669.1),camelpox (nucleotides 43331-43480 of GenBank Accession No. AY009089.1),ectromelia (nucleotides 51008-51157 of GenBank Accession No.AF012825.2), monkeypox (nucleotides 42515-42660 of GenBank Accession No.AF380138.1), and variola viruses (nucleotides 33100-33249 of GenBankAccession No. X69198.1). Accordingly, also provided are modifications ofthe equivalent of the F3 gene in poxviruses, such as orthopoxvirusesincluding a variety of vaccinia virus strains. One skilled in the artcan identify the location of the equivalent F3 gene in a variety ofpoxviruses, orthopoxviruses and vaccinia viruses. For example, anequivalent of the F3 gene in poxviruses, orthopoxviruses and vacciniaviruses can include a gene that contains at least 80%, at least 85%, atleast 90%, at least 92%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity with the nucleotidesequence of the F3 gene in SEQ ID NO:1. In another example, anequivalent of the F3 gene in poxviruses, orthopoxviruses and vacciniaviruses can include a gene that contains at least 80%, at least 85%, atleast 90%, at least 92%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity with the amino acidsequence of F3 in SEQ ID NO:2. In another example, the equivalent to theF3 gene in LIVP can be determined by its structural location in theviral genome: the F3 gene is located on the HindIII-F fragment ofvaccinia virus between open reading frames F14L and F15L as defined byGoebel et al., Virology (1990) 179:247-266, and in the oppositeorientation of ORFs F14L and F15L; one skilled in the art can readilyidentify the gene located in the structurally equivalent region in alarge variety of related viruses, such as a large variety of poxviruses.

Comparative protein sequence analysis revealed some insight into proteinfunction. The closest match with the protein encoded by the F3 gene(strain LIVP) is a prolyl 4-hydroxylase alpha subunit precursor (4-PHalpha) from the nematode Caenorhabditis elegans (Veijola et al., J.Biol. Chem. 269 (1994), 26746-26753). This alpha subunit forms an activealpha-beta dimer with the human protein disulfide isomerase betasubunit. Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of4-hydroxyproline in collagen. The vertebrate enzyme is an alpha 2-beta 2tetramer, the beta subunit of which is identical to the proteindisulfide-isomerase (PDI). The importance of this protein for vacciniaviral replication is unknown, but a deficiency of this protein canresult in retargeting vaccinia virus to tumor tissue.

d. Multiple Modifications

The vaccinia viruses provided herein also can contain two or moreinsertions, mutations or deletions. Thus, included are vaccinia virusescontaining two or more insertions, mutations or deletions of the lociprovided herein or other loci known in the art. In one embodiment, avaccinia virus contains an insertion, mutation or deletion in the F3gene, and one or more additional insertions, mutations or deletions. Inone embodiment of the modified vaccinia virus, at least the F3 gene hasbeen modified by insertion of a foreign nucleotide sequence.Modifications such as modification of the F3 gene will typically resultin at least partial inactivation of the gene or gene product. In oneexample, the F3 gene and the TK gene have been modified by insertion ofa foreign nucleotide sequence. In another example, the F3 gene and theHA gene have been modified by insertion of a foreign nucleotidesequence. In another example, the F3 gene and both the TK and HA geneshave been modified by insertion of a foreign nucleotide sequence. Inanother example, the HA gene and the TK gene have been modified byinsertion of a foreign nucleotide sequence. Accordingly, the presentcompositions and methods include a modified vaccinia virus wherein twoor more of (a) the F3 gene, (b) the TK gene, and (c) the HA gene havebeen modified. In one embodiment, at least two of the F3 gene, TK geneand HA gene have been inactivated, for example by insertion, deletionand/or replacement of nucleotide(s) within the coding region, orregulatory sequences of two or more of these genes have been inactivatedby insertion, deletion or mutation.

e. The Lister Strain

In another embodiment, the viruses and methods provided herein can bebased on modifications to the Lister strain of vaccinia virus. Lister(also referred to as Elstree) vaccinia virus is available from any of avariety of sources. For example, the Elstree vaccinia virus is availableat the ATCC under Accession Number VR-1549. The Lister vaccinia strainhas high transduction efficiency in tumor cells with high levels of geneexpression.

In one embodiment, the Lister strain can be an attenuated Lister strain,such as the LIVP (Lister virus from the Institute of Viral Preparations,Moscow, Russia) strain, which was produced by further attenuation of theLister strain. The LIVP strain was used for vaccination throughout theworld, particularly in India and Russia, and is widely available.

The LIVP strain has a reduced pathogenicity while maintaining a hightransduction efficiency. For example, as provided herein, F3-interruptedmodified LIVP vaccinia virus can selectively replicate in tumor cells invivo. In one embodiment, provided herein are modified LIVP viruses,including viruses having a modified TK gene, viruses having a modifiedHA gene, viruses having a modified F3 gene, and viruses having two ormore of: modified HA gene, modified TK gene, and modified F3 gene.

ii. Other Cytoplasmic Viruses

Also provided herein are cytoplasmic viruses that are not poxviruses.

Cytoplasmic viruses can replicate without introducing viral nucleic acidmolecules into the nucleus of the host cell. A variety of suchcytoplasmic viruses are known in the art, and include African swine flufamily viruses and various RNA viruses such as arenaviruses,picornaviruses, caliciviruses, togaviruses, coronaviruses,paramyxoviruses, flaviviruses, reoviruses, and rhaboviruses. Exemplarytogaviruses include Sindbis viruses. Exemplary arenaviruses includelymphocytic choriomeningitis virus. Exemplary rhaboviruses includevesicular stomatitis viruses. Exemplary paramyxo viruses includeNewcastle Disease viruses and measles viruses. Exemplary picornavirusesinclude polio viruses, bovine enteroviruses and rhinoviruses. Exemplaryflaviviruses include Yellow fever virus; attenuated Yellow fever virusesare known in the art, as exemplified in Barrett et al., Biologicals25:17-25 (1997), and McAllister et al., J. Virol. 74:9197-9205 (2000).

Also provided herein are modifications of the viruses provided above toenhance one or more characteristics relative to the wild type virus.Such characteristics can include, but are not limited to, attenuatedpathogenicity, reduced toxicity, preferential accumulation in tumor,increased ability to activate an immune response against tumor cells,increased immunogenicity, increased or decreased replication competence,and are able to express exogenous proteins, and combinations thereof. Insome embodiments, the modified viruses have an ability to activate animmune response against tumor cells without aggressively killing thetumor cells. In other embodiments, the viruses can be modified toexpress one or more detectable genes, including genes that can be usedfor imaging. In other embodiments, the viruses can be modified toexpress one or more genes for harvesting the gene products and/or forharvesting antibodies against the gene products.

b. Adenovirus, Herpes, Retroviruses

Further provided herein are viruses that include in their life cycleentry of a nucleic acid molecule into the nucleus of the host cell. Avariety of such viruses are known in the art, and include herpesviruses,papovaviruses, retroviruses, adenoviruses, parvoviruses andorthomyxoviruses. Exemplary herpesviruses include herpes simplex type 1viruses, cytomegaloviruses, and Epstein-Barr viruses. Exemplarypapovaviruses include human papillomavirus and SV40 viruses. Exemplaryretroviruses include lentiviruses. Exemplary orthomyxoviruses includeinfluenza viruses. Exemplary parvoviruses include adeno associatedviruses.

Also provided herein are modifications of the viruses provided above toenhance one or more characteristics relative to the wild type virus.Such characteristics can include, but are not limited to, attenuatedpathogenicity, reduced toxicity, preferential accumulation in tumor,increased ability to activate an immune response against tumor cells,increased immunogenicity, increased or decreased replication competence,and are able to express exogenous proteins, and combinations thereof. Insome embodiments, the modified viruses have an ability to activate animmune response against tumor cells without aggressively killing thetumor cells. In other embodiments, the viruses can be modified toexpress one or more detectable genes, including genes that can be usedfor imaging. In other embodiments, the viruses can be modified toexpress one or more genes for harvesting the gene products and/or forharvesting antibodies against the gene products.

3. Bacteria

Bacteria can also be used in the methods provided herein. Any of avariety of bacteria possessing the desired characteristics can be used.In one embodiment, aerobic bacteria can be used. In another embodiment,anaerobic bacteria can be used. In another embodiment, extracellularbacteria can be used. In another embodiment, intracellular bacteria canbe used.

In some embodiments, the bacteria provided herein can be extracellularbacteria. A variety of extracellular bacteria are known in the art andinclude vibrio, lactobacillus, streptococcus, escherichia. Exemplarybacteria include Vibrio cholerae, Streptococcus pyogenes, andEscherichia coli. In other embodiments, the bacteria provided herein canbe intracellular bacteria. A variety of intracellular bacteria are knownin the art and include listeria, salmonella, clostridium, andbifodobacterium. Exemplary intracellular bacteria include Listeriamonocytogenes, Salmonella typhimurium, Clostridium histolyticus,Clostridium butyricum, Bifodobacterium longum, and Bifodobacteriumadolescentis. Additional bacteria include plant bacteria such asClavibacter michiganensis subsp. michiganensis, Agrobacteriumtumefaciens, Erwinia herbicola, Azorhizobium caulinodans, Xanthomonascampestris pv. vesicatoria, and Xanthomonas campestris pv. campestris.

A further example of a bacteria provided herein are magnetic bacteria.Such bacteria allow tumor detection through the accumulation ofiron-based contrast agents. Magnetic bacteria can be isolated from freshand marine sediments. Magnetic bacteria can produce magnetic particles(Fe304) (Blakemore, Annu. Rev. Microbiol. 36 (1982), 217-238). To do so,the magnetic bacteria have efficient iron uptake systems, which allowthem to utilize both insoluble and soluble forms of iron.Magnetospirillum magnetic AMB-1 is an example of such magnetic bacteriathat has been isolated and cultured for magnetic particle production(Yang et al., Enzyme Microb. Technol. 29 (2001), 13-19). As providedherein, these magnetic bacteria (naturally occurring or geneticallymodified), when injected intravenously, can selectively accumulate intumor. Accordingly, these bacteria can be used for accumulatingiron-based contrast agents in the tumors, which in turn allows tumordetection by MRI. Similarly, other naturally isolated metal accumulatingstrains of bacteria can be used for tumor targeting, absorption ofmetals from contrast agents, and tumor imaging.

Also provided herein are modifications of bacteria to enhance one ormore characteristics relative to the wild type bacteria. Suchcharacteristics can include, but are not limited to, attenuatedpathogenicity, reduced toxicity, preferential accumulation in tumor,increased ability to activate an immune response against tumor cells,increased immunogenicity, increased or decreased replication competence,and are able to express exogenous proteins, and combinations thereof. Insome embodiments, the modified bacteria have an ability to activate animmune response against tumor cells without aggressively killing thetumor cells. In other embodiments, the bacteria can be modified toexpress one or more detectable genes, including genes that can be usedfor imaging. In other embodiments, the bacteria can be modified toexpress one or more genes for harvesting the gene products and/or forharvesting antibodies against the gene products.

a. Aerobic Bacteria

Previous studies have postulated that anaerobic bacteria are preferredfor administration to tumors (Lemmon et al., 1997 Gene Therapy4:791-796). As provided herein, it has been determined that aerobicbacteria can survive and grow in tumors. Accordingly, a bacteria used inthe methods provided herein can include a bacteria that can survive andgrow in an oxygenated environment. In some embodiments, the bacteriamust be in an oxygenated environment in order to survive and grow. Avariety of aerobic bacteria are known in the art, includinglactobacilli, salmonella, streptococci, staphylococci, vibrio, listeria,and escherichia. Exemplary bacteria include Vibrio cholerae, Listeriamonocytogenes, Salmonella typhimurium, Streptococcus pyogenes,Escherichia coli, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus paracasei,Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillussalivarius, Lactobacillus sporogenes, Lactobacillus lactis,Lactobacillus fermentum, Streptococcus thermophilus, Bacillus subtilis,Bacillus megaterium, Bacillus polymyxa, Myobacterium smegmatis,Mycobacterium vaccae, Mycobacterium microti, Mycobacterium habana,Enterococcus faecalis, Pseudomonas fluorescens, and Pseudomonas putida.

b. Anaerobic Bacteria

A bacteria used in the methods provided herein can include a bacteriathat does not require oxygen to survive and grow. In some embodiments,the bacteria must be in an oxygen-free environment in order to surviveand grow. A variety of aerobic bacteria are known in the art, includingclostridium, bifodobacterium. Exemplary bacteria include Clostridiumhistolyticus, Clostridium butyricum, Clostridium novyi, Clostridiumsordellii, Clostridium absonum, Clostridium bifermentans, Clostridiumdifficile, Clostridium histolyticum, Clostridium perfringens,Clostridium beijerinckii, Clostridium sporogenes, Staphylococcus aureus,Staphylococcus epidermidis, Bifidobacterium longum, Bifidobacteriumadolescentis, Bifidobacterium bifidum, Bifidobacterium infantis,Bifidobacterium laterosporus, Bifidobacterium animalis, Actinomycesisraelii, Eubacterium lentum, Peptostreptococcus anaerobis, Peptococcusprevotti, and Acidaminococcus fermentans.

4. Eukaryotic Cells

Also encompassed within the microorganisms provided herein and themethods of making and using such microorganisms are eukaryotic cells,including cells from multicellular eukaryotes, including mammals such asprimates, where exemplary cells are human cells. Typically the cells areisolated cells. For example, eukaryotic cells can be tumor cells,including mammalian tumor cells such as primate tumor cells, whereexemplary primate tumor cells are human tumor cells such as human breastcancer cells. In another example, eukaryotic cells can includefibrosarcoma cells such as human fibrosarcoma cells. Exemplary humanfibrosarcoma cells include HT1080 (ATCC Accession Nos. CCL-121,CRL-12011 or CRL-12012). In another example, eukaryotic cells caninclude stem cells, including mammalian stem cells such as primate stemcells, where exemplary primate stem cells are human stem cells.

Also provided herein are modifications of eukaryotic cells to enhanceone or more characteristics relative to the wild type cells. Suchcharacteristics can include, but are not limited to, attenuatedpathogenicity, reduced toxicity, preferential accumulation in tumor,increased ability to activate an immune response against tumor cells,increased immunogenicity, increased or decreased replication competence,and are able to express exogenous proteins, and combinations thereof. Insome embodiments, the modified eukaryotic cells have an ability toactivate an immune response against tumor cells without aggressivelykilling the tumor cells. In other embodiments, the eukaryotic cells canbe modified to express one or more detectable genes, including genesthat can be used for imaging. In other embodiments, the eukaryotic cellscan be modified to express one or more genes for harvesting the geneproducts and/or for harvesting antibodies against the gene products.

C. METHODS FOR MAKING A MODIFIED MICROORGANISM

The microorganisms provided herein can be formed by standardmethodologies well known in the art for modifying microorganisms such asviruses, bacteria and eukaryotic cells. Briefly, the methods includeintroducing into microorganisms one or more genetic modification,followed by screening the microorganisms for properties reflective ofthe modification or for other desired properties.

1. Genetic Modifications

Standard techniques in molecular biology can be used to generate themodified microorganisms provided herein. Such techniques include variousnucleic acid manipulation techniques, nucleic acid transfer protocols,nucleic acid amplification protocols, and other molecular biologytechniques known in the art. For example, point mutations can beintroduced into a gene of interest through the use of oligonucleotidemediated site-directed mutagenesis. Alternatively, homologousrecombination can be used to introduce a mutation or exogenous sequenceinto a target sequence of interest. Nucleic acid transfer protocolsinclude calcium chloride transformation/transfection, electroporation,liposome mediated nucleic acid transfer,N-[1-(2,3-Dioloyloxy)propyl]-N,N,N-trimethylammonium methylsulfatemeditated transformation, and others. In an alternative mutagenesisprotocol, point mutations in a particular gene can also be selected forusing a positive selection pressure. See, e.g., Current Techniques inMolecular Biology, (Ed. Ausubel, et al.). Nucleic acid amplificationprotocols include but are not limited to the polymerase chain reaction(PCR). Use of nucleic acid tools such as plasmids, vectors, promotersand other regulating sequences, are well known in the art for a largevariety of viruses and cellular organisms. Further a large variety ofnucleic acid tools are available from many different sources includingATCC, and various commercial sources. One skilled in the art will bereadily able to select the appropriate tools and methods for geneticmodifications of any particular virus or cellular organism according tothe knowledge in the art and design choice.

Any of a variety of modifications can be readily accomplished usingstandard molecular biological methods known in the art. Themodifications will typically be one or more truncations, deletions,mutations or insertions of the microorganismal genome. In oneembodiment, the modification can be specifically directed to aparticular sequence. The modifications can be directed to any of avariety of regions of the microorganismal genome, including, but notlimited to, a regulatory sequence, to a gene-encoding sequence, or to asequence without a known role. Any of a variety of regions ofmicroorganismal genomes that are available for modification are readilyknown in the art for many microorganisms, including the microorganismsspecifically listed herein. As a non-limiting example, the loci of avariety of vaccinia genes provided hereinelsewhere exemplify the numberof different regions that can be targeted for modification in themicroorganisms provided herein. In another embodiment, the modificationcan be fully or partially random, whereupon selection of any particularmodified microorganism can be determined according to the desiredproperties of the modified the microorganism.

In some embodiments, the microorganism can be modified to express anexogenous gene. Exemplary exogenous gene products include proteins andRNA molecules. The modified microorganisms can express a detectable geneproduct, a therapeutic gene product, a gene product for manufacturing orharvesting, or an antigenic gene product for antibody harvesting. Thecharacteristics of such gene products are described hereinelsewhere. Insome embodiments of modifying an organism to express an exogenous gene,the modification can also contain one or more regulatory sequences toregulate expression of the exogenous gene. As is known in the art,regulatory sequences can permit constitutive expression of the exogenousgene or can permit inducible expression of the exogenous gene. Further,the regulatory sequence can permit control of the level of expression ofthe exogenous gene. In some examples, inducible expression can be underthe control of cellular or other factors present in a tumor cell orpresent in a microorganism-infected tumor cell. In other examples,inducible expression can be under the control of an administerablesubstance, including IPTG, RU486 or other known induction compounds. Anyof a variety of regulatory sequences are available to one skilled in theart according to known factors and design preferences. In someembodiments, such as gene product manufacture and harvesting, theregulatory sequence can result in constitutive, high levels of geneexpression. In some embodiments, such as anti-(gene product) antibodyharvesting, the regulatory sequence can result in constitutive, lowerlevels of gene expression. In tumor therapy embodiments, a therapeuticprotein can be under the control of an internally inducible promoter oran externally inducible promoter.

In other embodiments, organ or tissue-specific expression can becontrolled by regulatory sequences. In order to achieve expression onlyin the target organ, for example, a tumor to be treated, the foreignnucleotide sequence can be linked to a tissue specific promoter and usedfor gene therapy. Such promoters are well known to those skilled in theart (see e.g., Zimmermann et al., (1994) Neuron 12, 11-24; Vidal et al.;(1990) EMBO J. 2, 833-840; Mayford et al., (1995), Cell 81, 891-904;Pinkert et al., (1987) Genes & Dev. 1, 268-76).

In some embodiments, the microorganisms can be modified to express twoor more proteins, where any combination of the two or more proteins canbe one or more detectable gene products, therapeutic gene products, geneproducts for manufacturing or harvesting, or antigenic gene products forantibody harvesting. In one embodiment, a microorganism can be modifiedto express a detectable protein and a therapeutic protein. In anotherembodiment, a microorganism can be modified to express two or more geneproducts for detection or two or more therapeutic gene products. Forexample, one or more proteins involved in biosynthesis of a luciferasesubstrate can be expressed along with luciferase. When two or moreexogenous genes are introduced, the genes can be regulated under thesame or different regulatory sequences, and the genes can be inserted inthe same or different regions of the microorganismal genome, in a singleor a plurality of genetic manipulation steps. In some embodiments, onegene, such as a gene encoding a detectable gene product, can be underthe control of a constitutive promoter, while a second gene, such as agene encoding a therapeutic gene product, can be under the control of aninducible promoter. Methods for inserting two or more genes in to amicroorganism are known in the art and can be readily performed for awide variety of microorganisms using a wide variety of exogenous genes,regulatory sequences, and/or other nucleic acid sequences.

In an example of performing microorganismal modification methods,vaccinia virus strain LIVP was modified to contain insertions ofexogenous DNA in three different locations of the viral genome. Usinggeneral methods known in the art, known molecular biology tools, andsequences known in the art or disclosed herein can be used to createmodified vaccinia virus strains, including viruses containing insertionsin the F3 gene, TK gene and/or HA gene. See, e.g., Mikryukov, et al.,Biotekhnologya 4 (1998), 442-449; Goebel et al., Virology 179 (1990),247-266; Antoine et al., Virology 244 (1998), 365-396; Mayr et al.,Zentbl. Bakteriol. Hyg. Abt 1 Orig. B 167 (1978), 375-390; Ando andMatumoto, Jpn. J. Microbial. 14 (1979), 181-186; Sugimoto et al.,Microbial. Immuol. 29 (1985), 421-428; Takahashi-Nishimaki et al., J.Gen. Virol. 68 (1987), 2705-2710). These methods include, for example,in vitro recombination techniques, synthetic methods and in vivorecombination methods as described, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory Press, cold Spring Harbor N.Y. (1989), and in the Examplesdisclosed herein. The person skilled in the art can isolate the geneencoding the gene product of F3 (or a related gene product) from anyvaccinia strain using, for example, the nucleotide sequence of the F3gene of SEQ ID NO:1 or SEQ ID NOS: 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30 or 32, or a fragment thereof as a probe for screening a library.

Methods of producing recombinant microorganisms are known in the art.Provided herein for exemplary purposes are methods of producing arecombinant vaccinia virus. A recombinant vaccinia virus with aninsertion in the F3 gene (NotI site of LIVP) can be prepared by thefollowing steps: (a) generating (i) a vaccinia shuttle plasmidcontaining the modified F3 gene inserted at restriction site X and (ii)a dephosphorylated wt VV (VGL) DNA digested at restriction site X; (b)transfecting host cells infected with PUV-inactivated helper VV (VGL)with a mixture of the constructs of (i) and (ii) of step a; and (c)isolating the recombinant vaccinia viruses from the transfectants. Oneskilled in the art knows how to perform such methods, for example byfollowing the instructions given in Example 1 and the legend to FIG. 1;see also Timiryasova et al., Biotechniques 31 (2001), 534-540. In oneembodiment, restriction site X is a unique restriction site. A varietyof suitable host cells also are known to the person skilled in the artand include many mammalian, avian and insect cells and tissues which aresusceptible for vaccinia virus infection, including chicken embryo,rabbit, hamster and monkey kidney cells, for example, HeLa cells, RK₁₃,CV-1, Vero, BSC40 and BSC-1 monkey kidney cells.

2. Screening for Above Characteristics

Modified microorganisms can be screened for any desired characteristics,including the characteristics described herein such as attenuatedpathogenicity, reduced toxicity, preferential accumulation in tumor,increased ability to activate an immune response against tumor cells,increased immunogenicity, increased or decreased replication competence,and are able to express exogenous proteins, and combinations thereof.For example, the modified microorganisms can be screened for the abilityto activate an immune response against tumor cells without aggressivelykilling the tumor cells. In another example, the microorganisms can bescreened for expression of one or more detectable genes, including genesthat can be used for imaging, or for expression of one or more genes formanufacture or harvest of the gene products and/or for harvest ofantibodies against the gene products.

Any of a variety of known methods for screening for such characteristicscan be performed, as demonstrated in the Examples provided herein. OneExemplary method for screening for desired characteristics includes, butis not limited to, monitoring growth, replication and/or gene expression(including expression of an exogenous gene) in cell culture or other invitro medium. The cell culture can be from any organism, and from anytissue source, and can include tumorous tissues. Other exemplary methodsfor screening for desired characteristics include, but are not limitedto, administering a microorganism to animal, including non-human animalssuch as a mouse, monkey or ape, and optionally also including humans,and monitoring the microorganism, the tumor, and or the animal;monitoring can be performed by in vivo imaging of the microorganismand/or the tumor (e.g., low light imaging of microorganismal geneexpression or ultrasonic tumor imaging), external monitoring of thetumor (e.g., external measurement of tumor size), monitoring the animal(e.g., monitoring animal weight, blood panel, antibody titer, spleensize, or liver size). Other exemplary methods for screening for desiredcharacteristics include, but are not limited to, harvesting a non-humananimal for the effects and location of the microorganism and expressionby the microorganism, including methods such as harvesting a variety oforgans including a tumor to determine presence of the microorganismand/or gene expression by the microorganism in the organs or tumor,harvesting of organs associated with an immune response ormicroorganismal clearance such as the spleen or liver, harvesting thetumor to determine tumor size and viability of tumor cells, harvestingantibodies or antibody producing cells. Such screening and monitoringmethods can be used in any of a variety of combinations, as is known inart. In one embodiment, a microorganism can be screened by administeringthe microorganism to an animal such as a non-human animal or a human,followed by monitoring by in vivo imaging. In another embodiment, amicroorganism can be screened by administering the microorganism to ananimal such as a non-human animal, monitoring by in vivo imaging, andthen harvesting the animal. Thus, provided herein are methods forscreening a microorganism for desired characteristics by administeringthe microorganism to an animal such as an animal with a tumor, andmonitoring the animal, tumor (if present), and/or microorganism in theanimal for one or more characteristics. Also provided herein are methodsfor screening a microorganism for desired characteristics byadministering the microorganism to a non-human animal such as anon-human animal with a tumor, harvesting the animal, and assaying theanimal's organs, antibody titer, and/or tumor (if present) for one ormore characteristics.

Provided herein are methods for screening a microorganism for attenuatedpathogenicity or reduced toxicity, where the pathogenicity or toxicitycan be determined by a variety of techniques, including, but not limitedto, assessing the health state of the subject, measuring the body weightof a subject, blood or urine analysis of a subject, and monitoringtissue distribution of the microorganism within the subject; suchtechniques can be performed on a living subject in vivo, or can beperformed post mortem. Methods also can include the ability of themicroorganisms to lyse cells or cause cell death, which can bedetermined in vivo or in vitro.

When a subject drops below a threshold body weight, the microorganismcan be considered pathogenic to the subject. Exemplary thresholds can bea drop of about 5% or more, a drop of about 10% or more, or a drop ofabout 15% or more in body weight relative to a reference. A body weightreference can be selected from any of a variety of references used inthe art; for example, a body weight reference can be the weight of thesubject prior to administration of the microorganism, the body weightreference can be a control subject having the same condition as the testsubject (e.g., normal or tumor-injected), where the change in weight ofthe control is compared to the change in weight of the test subject forthe time period after administration of the microorganism.

Blood or urine analysis of the subject can indicate level of immuneresponse, level of toxins in the subject, or other levels of stress tocells, tissues or organs of the subject such as kidneys, pancreas, liverand spleen. Levels increased above established threshold levels canindicate pathogenicity of the microorganism to the subject. Thresholdlevels of components of blood or urine for indicating microorganismalpathogenicity are well known in the art, and any such thresholds can beselected herein according to the desired tolerance of pathogenicity ortoxicity of the microorganism.

Tissue distribution of a microorganism in a subject can indicatepathogenicity or toxicity of the microorganism. In one embodiment,tissue distribution of a microorganism that is not pathogenic or toxiccan be mostly in tumor relative to other tissues or organs.Microorganisms located mostly in tumor can accumulate, for example, atleast about 2-fold greater, at least about 5-fold greater, at leastabout 10-fold greater, at least about 100-fold greater, at least about1,000-fold greater, at least about 10,000-fold greater, at least about100,000-fold greater, or at least about 1,000,000-fold greater, than themicroorganisms that accumulate in any other particular organ or tissue.

Provided herein are methods for screening a microorganism for tissuedistribution or accumulation, where the tissue distribution can bedetermined by a variety of techniques, including, but not limited to,harvesting a non-human subject, in vivo imaging a detectable geneproduct in subject. Harvesting can be accomplished by euthanizing thenon-human subject, and determining the accumulation of microorganisms intumor and, optionally, the accumulation in one or more additionaltissues or organs. The accumulation can be determined by any of avariety of methods, including, but not limited to, detecting geneproducts such as detectable gene products (e.g., gfp or betagalactosidase), histological or microscopic evaluation of tissue, organor tumor samples, or measuring the number of plaque or colony formingunits present in a tissue, organ or tumor sample. In one embodiment, thedesired amount of tissue distribution of a microorganism can be mostlyin tumor relative to other tissues or organs. Microorganisms locatedmostly in tumor can accumulate, for example, at least about 2-foldgreater, at least about 5-fold greater, at least about 10-fold greater,at least about 100-fold greater, at least about 1,000-fold greater, atleast about 10,000-fold greater, at least about 100,000-fold greater, orat least about 1,000,000-fold greater, than the microorganisms thataccumulate in any other particular organ or tissue.

Also provided herein are methods of screening for microorganisms thatcan elicit an immune response, where the immune response can be againstthe tumor cells or against the microorganisms. A variety of methods formeasuring the ability to elicit an immune response are known in the art,and include measuring an overall increase in immune activity in asubject, measuring an increase in anti-microorganism or anti-tumorantibodies in a subject, testing the ability of a microorganism-treated(typically a non-human) subject to prevent later infection/tumorformation or to rapidly eliminate microorganisms or tumor cells. Methodsalso can include the ability of the microorganisms to lyse cells orcause cell death, which can be determined in vivo or in vitro.

Also provided herein are methods for determining increased or decreasedreplication competence, by monitoring the speed of replication of themicroorganisms. Such measurements can be performed in vivo or in vitro.For example, the speed of replication in a cell culture can be used todetermine replication competence of a microorganism. In another example,the speed of replication in a tissue, organ or tumor in a subject can beused to measure replication competence. In some embodiments, decreasedreplication competence in non-tumor tissues and organs can be thecharacteristic to be selected in a screen. In other embodiments,increased replication competence in tumors can be the characteristic tobe selected in a screen.

Also provided herein are methods for determining the ability of amicroorganism to express genes, such as exogenous genes. Such methodscan be performed in vivo or in vitro. For example, the microorganismscan be screened on selective plates for the ability to express a genethat permits survival of the microorganism or permits the microorganismto provide a detectable signal, such as turning X-gal blue. Such methodsalso can be performed in vivo, where expression can be determined, forexample, by harvesting tissues, organs or tumors a non-human subject orby in vivo imaging of a subject.

Also provided herein are methods for determining the ability of amicroorganism to express genes toward which the subject can developantibodies, including exogenous genes toward which the subject candevelop antibodies. Such methods can be performed in vivo using any of avariety of non-human subjects. For example, gene expression can bedetermined, for example, by bleeding a non-human subject to which amicroorganism has been administered, and assaying the blood (or serum)for the presence of antibodies against the microorganism-expressed gene,or by any other method generally used for polyclonal antibodyharvesting, such as production bleeds and terminal bleeds.

Also provided herein are methods for screening a microorganism that hastwo or more characteristics provided herein, including screening forattenuated pathogenicity, reduced toxicity, preferential accumulation intumor, increased ability to activate an immune response against tumorcells, increased immunogenicity, increased or decreased replicationcompetence, ability to express exogenous proteins, and ability to elicitantibody production against a microorganismally expressed gene product.A single monitoring technique, such as in vivo imaging, can be used toverify two or more characteristics, or a variety of different monitoringtechniques can be used, as can be determined by one skilled in the artaccording to the selected characteristics and according to themonitoring techniques used.

D. THERAPEUTIC METHODS

Provided herein are therapeutic methods, including methods of treatingor preventing immunoprivileged cells or tissue, including cancerouscells, tumor and metastasis. The methods provided herein includeadministering a microorganism to a subject containing a tumor and/ormetastases. The methods provided herein do not require the microorganismto kill tumor cells or decrease the tumor size. Instead, the methodsprovided herein include administering to a subject a microorganism thatcan cause or enhance an anti-tumor immune response in the subject. Insome embodiments, the microorganisms provided herein can be administeredto a subject without causing microorganism-induced disease in thesubject. In some embodiments, the microorganisms can accumulate intumors or metastases. In some embodiments, the microorganisms can elicitan anti-tumor immune response in the subject, where typically themicroorganism-mediated anti-tumor immune response can develop overseveral days, such as a week or more, 10 days or more, two weeks ormore, or a month or more, as a result of little or nomicroorganism-cause tumor cell death. In some exemplary methods, themicroorganism can be present in the tumor, and can cause an anti-tumorimmune response without the microorganism itself causing enough tumorcell death to prevent tumor growth.

In some embodiments, provided herein are methods for eliciting orenhancing antibody production against a selected antigen or a selectedantigen type in a subject, where the methods include administering to asubject a microorganism that can accumulate in a tumor and/ormetastasis, and can cause release of a selected antigen or selectedantigen type from the tumor, resulting in antibody production againstthe selected antigen or selected antigen type. The administeredmicroorganisms can posses one or more characteristics includingattenuated pathogenicity, low toxicity, preferential accumulation intumor, ability to activate an immune response against tumor cells,immunogenicity, replication competence, ability to express exogenousgenes, and ability to elicit antibody production against amicroorganismally expressed gene product.

Any of a variety of antigens can be targeted in the methods providedherein, including a selected antigen such as an exogenous gene productexpressed by the microorganism, or a selected antigen type such as oneor more tumor antigens release from the tumor as a result ofmicroorganism infection of the tumor (e.g., by lysis, apoptosis,secretion or other mechanism of causing antigen release from the tumor).In at least some embodiments, it can be desirable to maintain release ofthe selected antigen or selected antigen type over a series of days, forexample, at least a week, at least ten days, at least two weeks or atleast a month.

Also provided herein are methods for providing a sustained antigenrelease within a subject, where the methods include administering to asubject a microorganism that can accumulate in a tumor and/ormetastasis, and can cause sustained release of an antigen, resulting inantibody production against the antigen. The sustained release ofantigen can last for several days, for example, at least a week, atleast ten days, at least two weeks or at least a month. The administeredmicroorganisms can posses one or more characteristics includingattenuated pathogenicity, low toxicity, preferential accumulation intumor, ability to activate an immune response against tumor cells,immunogenicity, replication competence, ability to express exogenousgenes, and ability to elicit antibody production against amicroorganismally expressed gene product. The sustained release ofantigen can result in an immune response by the microorganism-infectedhost, in which the host can develop antibodies against the antigen,and/or the host can mount an immune response against cells expressingthe antigen, including an immune response against tumor cells. Thus, thesustained release of antigen can result in immunization against tumorcells. In some embodiments, the microorganism-mediated sustained antigenrelease-induced immune response against tumor cells can result incomplete removal or killing of all tumor cells.

Also provided herein are methods for inhibiting tumor growth in asubject, where the methods include administering to a subject amicroorganism that can accumulate in a tumor and/or metastasis, and cancause or enhance an anti-tumor immune response. The anti-tumor immuneresponse induced as a result of tumor or metastases-accumulatedmicroorganisms can result in inhibition of tumor growth. Theadministered microorganisms can posses one or more characteristicsincluding attenuated pathogenicity, low toxicity, preferentialaccumulation in tumor, ability to activate an immune response againsttumor cells, immunogenicity, replication competence, ability to expressexogenous genes, and ability to elicit antibody production against amicroorganismally expressed gene product.

Also provided herein are methods for inhibiting growth or formation of ametastasis in a subject, where the methods include administering to asubject a microorganism that can accumulate in a tumor and/ormetastasis, and can cause or enhance an anti-tumor immune response. Theanti-tumor immune response induced as a result of tumor ormetastasis-accumulated microorganisms can result in inhibition ofmetastasis growth or formation. The administered microorganisms canposses one or more characteristics including attenuated pathogenicity,low toxicity, preferential accumulation in tumor, ability to activate animmune response against tumor cells, immunogenicity, replicationcompetence, ability to express exogenous genes, and ability to elicitantibody production against a microorganismally expressed gene product.

Also provided herein are methods for decreasing the size of a tumorand/or metastasis in a subject, where the methods include administeringto a subject a microorganism that can accumulate in a tumor and/ormetastasis, and can cause or enhance an anti-tumor immune response. Theanti-tumor immune response induced as a result of tumor ormetastasis-accumulated microorganisms can result in a decrease in thesize of the tumor and/or metastasis. The administered microorganisms canposses one or more characteristics including attenuated pathogenicity,low toxicity, preferential accumulation in tumor, ability to activate animmune response against tumor cells, immunogenicity, replicationcompetence, ability to express exogenous genes, and ability to elicitantibody production against a microorganismally expressed gene product.

Also provided herein are methods for eliminating a tumor and/ormetastasis from a subject, where the methods include administering to asubject a microorganism that can accumulate in a tumor and/ormetastasis, and can cause or enhance an anti-tumor immune response. Theanti-tumor immune response induced as a result of tumor ormetastasis-accumulated microorganisms can result in elimination of thetumor and/or metastasis from the subject. The administeredmicroorganisms can posses one or more characteristics includingattenuated pathogenicity, low toxicity, preferential accumulation intumor, ability to activate an immune response against tumor cells,immunogenicity, replication competence, ability to express exogenousgenes, and ability to elicit antibody production against amicroorganismally expressed gene product.

Methods of reducing inhibiting tumor growth, inhibiting metastasisgrowth and/or formation, decreasing the size of a tumor or metastasis,eliminating a tumor or metastasis, or other tumor therapeutic methodsprovided herein include causing or enhancing an anti-tumor immuneresponse in the host. The immune response of the host, being anti-tumorin nature, can be mounted against tumors and/or metastases in whichmicroorganisms have accumulated, and can also be mounted against tumorsand/or metastases in which microorganisms have not accumulated,including tumors and/or metastases that form after administration of themicroorganisms to the subject. Accordingly, a tumor and/or metastasiswhose growth or formation is inhibited, or whose size is decreased, orthat is eliminated, can be a tumor and/or metastasis in which themicroorganisms have accumulated, or also can be a tumor and/ormetastasis in which the microorganisms have not accumulated.Accordingly, provided herein are methods of reducing inhibiting tumorgrowth, inhibiting metastasis growth and/or formation, decreasing thesize of a tumor or metastasis, eliminating a tumor or metastasis, orother tumor therapeutic methods, where the method includes administeringto a subject a microorganism, where the microorganism accumulates in atleast one tumor or metastasis and causes or enhances an anti-tumorimmune response in the subject, and the immune response also is mountedagainst a tumor and/or metastasis in which the microorganism cell didnot accumulate. In another embodiment, methods are provided forinhibiting or preventing recurrence of a neoplastic disease orinhibiting or preventing new tumor growth, where the methods includeadministering to a subject a microorganism that can accumulate in atumor and/or metastasis, and can cause or enhance an anti-tumor immuneresponse, and the anti-tumor immune response can inhibit or preventrecurrence of a neoplastic disease or inhibit or prevent new tumorgrowth.

The tumor or neoplastic disease therapeutic methods provided herein,such as methods of reducing inhibiting tumor growth, inhibitingmetastasis growth and/or formation, decreasing the size of a tumor ormetastasis, eliminating a tumor or metastasis, or other tumortherapeutic methods, also can include administering to a subject amicroorganism that can cause tumor cell lysis or tumor cell death. Sucha microorganism can be the same microorganism as the microorganism thatcan cause or enhance an anti-tumor immune response in the subject.Microorganisms, such as the microorganisms provided herein, can causecell lysis or tumor cell death as a result of expression of anendogenous gene or as a result of an exogenous gene. Endogenous orexogenous genes can cause tumor cell lysis or inhibit cell growth as aresult of direct or indirect actions, as is known in the art, includinglytic channel formation or activation of an apoptotic pathway. Geneproducts, such as exogenous gene products can function to activate aprodrug to an active, cytotoxic form, resulting in cell death where suchgenes are expressed.

Such methods of antigen production or tumor and/or metastasis treatmentcan include administration of a modified microorganism described hereinor a microorganism having modifications with a functional equivalence tothe vaccinia virus provided herein containing a modification of the F3gene and the TK gene and/or the HA gene, for therapy, such as for genetherapy, for cancer gene therapy, or for vaccine therapy. Such amicroorganism can be used to stimulate humoral and/or cellular immuneresponse, induce strong cytotoxic T lymphocytes responses in subjectswho may benefit from such responses. For example, the microorganism canprovide prophylactic and therapeutic effects against a tumor infected bythe microorganism or other infectious diseases, by rejection of cellsfrom tumors or lesions using microorganisms that express immunoreactiveantigens (Earl et al. (1986), Science 234, 728-831; Lathe et al. (1987),Nature (London) 326, 878-880), cellular tumor-associated antigens(Bernards et al., (1987), Proc. Natl. Acad. Sci. USA 84, 6854-6858;Estin et al. (1988), Proc. Natl. Acad. Sci. USA 85, 1052-1056; Kantor etal. (1992), J. Natl. Cancer Inst. 84, 1084-1091; Roth et al. (1996),Proc. Natl. Acad. Sci. USA 93, 4781-4786) and/or cytokines (e.g., IL-2,IL-12), costimulatory molecules (B7-1, B7-2) (Rao et al. (1996), J.Immunol. 156, 3357-3365; Chamberlain et al. (1996), Cancer Res. 56,2832-2836; Oertli et al. (1996), J. Gen. Virol. 77, 3121-3125; Qin andChatterjee (1996), Human Gene Ther. 2, 1853-1860; McAneny et al. (1996),Ann. Surg. Oncol. 3, 495-500), or other therapeutic proteins.

Provided herein, solid tumors can be treated with microorganisms, suchas vaccinia viruses, resulting in an enormous tumor-specificmicroorganism replication, which can lead to tumor protein antigen andviral protein production in the tumors. As provided herein, vacciniavirus administration to mice resulted in lysis of the infected tumorcells and a resultant release of tumor-cell-specific antigens.Continuous leakage of these antigens into the body led to a very highlevel of antibody titer (in approximately 7-14 days) against tumorproteins, viral proteins, and the virus encoded engineered proteins inthe mice. The newly synthesized antitumor antibodies and the enhancedmacrophage, neutrophils count were continuously delivered via thevasculature to the tumor and thereby provided for the recruitment of anactivated immune system against the tumor. The activated immune systemthen eliminated the foreign compounds of the tumor including the viralparticles. This interconnected release of foreign antigens boostedantibody production and continuous response of the antibodies againstthe tumor proteins to function like an autoimmunizing vaccination systeminitiated by vaccinia viral infection and replication, followed by celllysis, protein leakage and enhanced antibody production. Thus, thepresent methods can provide a complete process that can be applied toall tumor systems with immunoprivileged tumor sites as site ofprivileged viral, bacterial, and mammalian cell growth, which can leadto tumor elimination by the host's own immune system.

In other embodiments, methods are provided for immunizing a subject,where the methods include administering to the subject a microorganismthat expresses one or more antigens against which antigens the subjectwill develop an immune response. The immunizing antigens can beendogenous to the microorganism, such as vaccinia antigens on a vacciniavirus used to immunize against smallpox, or the immunizing antigens canbe exogenous antigens expressed by the microorganism, such as influenzaor HIV antigens expressed on a viral capsid or bacterial cell surface.Thus, the microorganisms provided herein, including the modifiedvaccinia viruses can be used as vaccines.

1. Administration

In performing the methods provided herein, a microorganism can beadministered to a subject, including a subject having a tumor or havingneoplastic cells, or a subject to be immunized. An administeredmicroorganism can be a microorganism provided herein or any othermicroorganism known for administration to a subject, for example, anyknown microorganism known for therapeutic administration to a subject,including antigenic microorganisms such as any microorganism known to beused for vaccination. In some embodiments, the microorganismadministered is a microorganism containing a characteristic such asattenuated pathogenicity, low toxicity, preferential accumulation intumor, ability to activate an immune response against tumor cells, highimmunogenicity, replication competence, and ability to express exogenousproteins, and combinations thereof.

a. Steps Prior to Administering the Microorganism

In some embodiments, one or more steps can be performed prior toadministration of the microorganism to the subject. Any of a variety ofpreceding steps can be performed, including, but not limited todiagnosing the subject with a condition appropriate for microorganismaladministration, determining the immunocompetence of the subject,immunizing the subject, treating the subject with a chemotherapeuticagent, treating the subject with radiation, or surgically treating thesubject.

For embodiments that include administering a microorganism to atumor-bearing subject for therapeutic purposes, the subject hastypically been previously diagnosed with a neoplastic condition.Diagnostic methods also can include determining the type of neoplasticcondition, determining the stage of the neoplastic conditions,determining the size of one or more tumors in the subject, determiningthe presence or absence of metastatic or neoplastic cells in the lymphnodes of the subject, or determining the presence of metastases of thesubject. Some embodiments of therapeutic methods for administering amicroorganism to a subject can include a step of determination of thesize of the primary tumor or the stage of the neoplastic disease, and ifthe size of the primary tumor is equal to or above a threshold volume,or if the stage of the neoplastic disease is at or above a thresholdstage, a microorganism is administered to the subject. In a similarembodiment, if the size of the primary tumor is below a thresholdvolume, or if the stage of the neoplastic disease is at or below athreshold stage, the microorganism is not yet administered to thesubject; such methods can include monitoring the subject until the tumorsize or neoplastic disease stage reaches a threshold amount, and thenadministering the microorganism to the subject. Threshold sizes can varyaccording to several factors, including rate of growth of the tumor,ability of the microorganism to infect a tumor, and immunocompetence ofthe subject. Generally the threshold size will be a size sufficient fora microorganism to accumulate and replicate in or near the tumor withoutbeing completely removed by the host's immune system, and will typicallyalso be a size sufficient to sustain a microorganismal infection for atime long enough for the host to mount an immune response against thetumor cells, typically about one week or more, about ten days or more,or about two weeks or more. Exemplary threshold tumor sizes for virusessuch as vaccinia viruses are at least about 100 mm³, at least about 200mm³, at least about 300 mm³, at least about 400 mm³, at least about 500mm³, at least about 750 mm³, at least about 1000 mm³, or at least about1500 mm³. Threshold neoplastic disease stages also can vary according toseveral factors, including specific requirement for staging a particularneoplastic disease, aggressiveness of growth of the neoplastic disease,ability of the microorganism to infect a tumor or metastasis, andimmunocompetence of the subject. Generally the threshold stage will be astage sufficient for a microorganism to accumulate and replicate in atumor or metastasis without being completely removed by the host'simmune system, and will typically also be a size sufficient to sustain amicroorganismal infection for a time long enough for the host to mountan immune response against the neoplastic cells, typically about oneweek or more, about ten days or more, or about two weeks or more.Exemplary threshold stages are any stage beyond the lowest stage (e.g.,Stage I or equivalent), or any stage where the primary tumor is largerthan a threshold size, or any stage where metastatic cells are detected.

In other embodiments, prior to administering to the subject amicroorganism, the immunocompetence of the subject can be determined.The methods of administering a microorganism to a subject providedherein can include causing or enhancing an immune response in a subject.Accordingly, prior to administering a microorganism to a subject, theability of a subject to mount an immune response can be determined. Anyof a variety of tests of immunocompetence known in the art can beperformed in the methods provided herein. Exemplary immunocompetencetests can examine ABO hemagglutination titers (IgM), leukocyte adhesiondeficiency (LAD), granulocyte function (NBT), T and B cell quantitation,tetanus antibody titers, salivary IgA, skin test, tonsil test,complement C3 levels, and factor B levels, and lymphocyte count. Oneskilled in the art can determine the desirability to administer amicroorganism to a subject according to the level of immunocompetence ofthe subject, according to the immunogenicity of the microorganism, and,optionally, according to the immunogenicity of the neoplastic disease tobe treated. Typically, a subject can be considered immunocompetent ifthe skilled artisan can determine that the subject is sufficientlycompetent to mount an immune response against the microorganism.

In some embodiments, the subject can be immunized prior to administeringto the subject a microorganism according to the methods provided herein.Immunization can serve to increase the ability of a subject to mount animmune response against the microorganism, or increase the speed atwhich the subject can mount an immune response against a microorganism.Immunization also can serve to decrease the risk to the subject ofpathogenicity of the microorganism. In some embodiments, theimmunization can be performed with an immunization microorganism that issimilar to the therapeutic microorganism to be administered. Forexample, the immunization microorganism can be a replication-incompetentvariant of the therapeutic microorganism. In other embodiments, theimmunization material can be digests of the therapeutic microorganism tobe administered. Any of a variety of methods for immunizing a subjectagainst a known microorganism are known in the art and can be usedherein. In one example, vaccinia viruses treated with, for example, 1microgram of psoralen and ultraviolet light at 365 nm for 4 minutes, canbe rendered replication incompetent. In another embodiment, themicroorganism can be selected as the same or similar to a microorganismagainst which the subject has been previously immunized, e.g., in achildhood vaccination.

In another embodiment, the subject can have administered thereto amicroorganism without any previous steps of cancer treatment such aschemotherapy, radiation therapy or surgical removal of a tumor and/ormetastases. The methods provided herein take advantage of the ability ofthe microorganisms to enter or localize near a tumor, where the tumorcells can be protected from the subject's immune system; themicroorganisms can then proliferate in such an immunoprotected regionand can also cause the release, typically a sustained release, of tumorantigens from the tumor to a location in which the subject's immunesystem can recognize the tumor antigens and mount an immune response. Insuch methods, existence of a tumor of sufficient size or sufficientlydeveloped immunoprotected state can be advantageous for successfuladministration of the microorganism to the tumor, and for sufficienttumor antigen production. If a tumor is surgically removed, themicroorganisms may not be able to localize to other neoplastic cells(e.g., small metastases) because such cells may not yet have maturedsufficiently to create an immunoprotective environment in which themicroorganisms can survive and proliferate, or even if themicroorganisms can localize to neoplastic cells, the number of cells orsize of the mass may be too small for the microorganisms to cause asustained release of tumor antigens in order for the host to mount ananti-tumor immune response. Thus, for example, provided herein aremethods of treating a tumor or neoplastic disease in whichmicroorganisms are administered to a subject with a tumor or neoplasticdisease without removing the primary tumor, or to a subject with a tumoror neoplastic disease in which at least some tumors or neoplastic cellsare intentionally permitted to remain in the subject. In other typicalcancer treatment methods such as chemotherapy or radiation therapy, suchmethods typically have a side effect of weakening the subject's immunesystem. This treatment of a subject by chemotherapy or radiation therapycan reduce the subject's ability to mount an anti-tumor immune response.Thus, for example, provided herein are methods of treating a tumor orneoplastic disease in which microorganisms are administered to a subjectwith a tumor or neoplastic disease without treating the subject with animmune system-weakening therapy, such as chemotherapy or radiationtherapy.

In an alternative embodiment, prior to administration of a microorganismto the subject, the subject can be treated in one or more cancertreatment steps that do not remove the primary tumor or that do notweaken the immune system of the subject. A variety of more sophisticatedcancer treatment methods are being developed in which the tumor can betreated without surgical removal or immune-system weakening therapy.Exemplary methods include administering a compound that decreases therate of proliferation of the tumor or neoplastic cells without weakeningthe immune system (e.g., by administering tumor suppressor compounds orby administering tumor cell-specific compounds) or administering anangiogenesis-inhibiting compound. Thus, combined methods that includeadministering a microorganism to a subject can further improve cancertherapy. Thus, provided herein are methods of administering amicroorganism to a subject, along with prior to or subsequent to, forexample, administering a compound that slows tumor growth withoutweakening the subject's immune system or a compound that inhibitsvascularization of the tumor.

b. Mode of Administration

Any mode of administration of a microorganism to a subject can be used,provided the mode of administration permits the microorganism to enter atumor or metastasis. Modes of administration can include, but are notlimited to, intravenous, intraperitoneal, subcutaneous, intramuscular,topical, intratumor, multipuncture (e.g., as used with smallpoxvaccines), inhalation, intranasal, oral, intracavity (e.g.,administering to the bladder via a catheter, administering to the gut bysuppository or enema), aural, or ocular administration. One skilled inthe art can select any mode of administration compatible with thesubject and the microorganism, and that also is likely to result in themicroorganism reaching tumors and/or metastases. The route ofadministration can be selected by one skilled in the art according toany of a variety of factors, including the nature of the disease, thekind of tumor, and the particular microorganism contained in thepharmaceutical composition. Administration to the target site can beperformed, for example, by ballistic delivery, as a colloidal dispersionsystem, or systemic administration can be performed by injection into anartery.

c. Dosage

The dosage regimen can be any of a variety of methods and amounts, andcan be determined by one skilled in the art according to known clinicalfactors. As is known in the medical arts, dosages for any one patientcan depend on many factors, including the subject's species, size, bodysurface area, age, sex, immunocompetence, and general health, theparticular microorganism to be administered, duration and route ofadministration, the kind and stage of the disease, for example, tumorsize, and other compounds such as drugs being administered concurrently.In addition to the above factors, such levels can be affected by theinfectivity of the microorganism, and the nature of the microorganism,as can be determined by one skilled in the art. At least some of theviruses used the in the methods provided herein can be more infectiousthan the bacteria used herein. Thus, in some embodiments of the presentmethods, virus can be administered at lower levels than bacteria. In thepresent methods, appropriate minimum dosage levels of microorganisms canbe levels sufficient for the microorganism to survive, grow andreplicate in a tumor or metastasis. Exemplary minimum levels foradministering a virus to a 65 kg human can include at least about 5×10⁵plaque forming units (pfu), at least about 1×10⁶ pfu, at least about5×10⁶ pfu, at least about 1×10⁷ pfu, or at least about 1×10⁸ pfu.Exemplary minimum levels for administering a bacterium to a 65 kg humancan include at least about 5×10⁶ colony forming units (cfu), at leastabout 1×10⁷ cfu, at least about 5×10⁷ cfu, at least about 1×10⁸ cfu, orat least about 1×10⁹ cfu. In the present methods, appropriate maximumdosage levels of microorganisms can be levels that are not toxic to thehost, levels that do not cause splenomegaly of 3× or more, levels thatdo not result in colonies or plaques in normal tissues or organs afterabout 1 day or after about 3 days or after about 7 days. Exemplarymaximum levels for administering a virus to a 65 kg human can include nomore than about 5×10¹⁰ pfu, no more than about 1×10¹⁰ pfu, no more thanabout 5×10⁹ pfu, no more than about 1×10⁹ pfu, or no more than about1×10⁸ pfu. Exemplary maximum levels for administering a bacterium to a65 kg human can include no more than about 5×10¹¹ pfu, no more thanabout 1×10¹¹ pfu, no more than about 5×10¹⁰ pfu, no more than about1×10¹⁰ pfu, or no more than about 1×10⁹ pfu.

d. Number of Administrations

The methods provided herein can include a single administration of amicroorganism to a subject or multiple administrations of amicroorganism to a subject. In some embodiments, a single administrationis sufficient to establish a microorganism in a tumor, where themicroorganism can proliferate and can cause or enhance an anti-tumorresponse in the subject; such methods do not require additionaladministrations of a microorganism in order to cause or enhance ananti-tumor response in a subject, which can result, for example ininhibition of tumor growth, inhibition of metastasis growth orformation, reduction in tumor or metastasis size, elimination of a tumoror metastasis, inhibition or prevention of recurrence of a neoplasticdisease or new tumor formation, or other cancer therapeutic effects. Inother embodiments, a microorganism can be administered on differentoccasions, separated in time typically by at least one day. Separateadministrations can increase the likelihood of delivering amicroorganism to a tumor or metastasis, where a previous administrationmay have been ineffective in delivering a microorganism to a tumor ormetastasis. Separate administrations can increase the locations on atumor or metastasis where microorganism proliferation can occur or canotherwise increase the titer of microorganism accumulated in the tumor,which can increase the scale of release of antigens or other compoundsfrom the tumor in eliciting or enhancing a host's anti-tumor immuneresponse, and also can, optionally, increase the level ofmicroorganism-based tumor lysis or tumor cell death. Separateadministrations of a microorganism can further extend a subject's immuneresponse against microorganismal antigens, which can extend the host'simmune response to tumors or metastases in which microorganisms haveaccumulated, and can increase the likelihood of a host mounting ananti-tumor immune response.

When separate administrations are performed, each administration can bea dosage amount that is the same or different relative to otheradministration dosage amounts. In one embodiment, all administrationdosage amounts are the same. In other embodiments, a first dosage amountcan be a larger dosage amount than one or more subsequent dosageamounts, for example, at least 10× larger, at least 100× larger, or atleast 1000× larger than subsequent dosage amounts. In one example of amethod of separate administrations in which the first dosage amount isgreater than one or more subsequent dosage amounts, all subsequentdosage amounts can be the same, smaller amount relative to the firstadministration.

Separate administrations can include any number of two or moreadministrations, including two, three, four, five or sixadministrations. One skilled in the art can readily determine the numberof administrations to perform or the desirability of performing one ormore additional administrations according to methods known in the artfor monitoring therapeutic methods and other monitoring methods providedherein. Accordingly, the methods provided herein include methods ofproviding to the subject one or more administrations of a microorganism,where the number of administrations can be determined by monitoring thesubject, and, based on the results of the monitoring, determiningwhether or not to provide one or more additional administrations.Deciding on whether or not to provide one or more additionaladministrations can be based on a variety of monitoring results,including, but not limited to, indication of tumor growth or inhibitionof tumor growth, appearance of new metastases or inhibition ofmetastasis, the subject's anti-microorganism antibody titer, thesubject's anti-tumor antibody titer, the overall health of the subject,the weight of the subject, the presence of microorganism solely in tumorand/or metastases, the presence of microorganism in normal tissues ororgans.

The time period between administrations can be any of a variety of timeperiods. The time period between administrations can be a function ofany of a variety of factors, including monitoring steps, as described inrelation to the number of administrations, the time period for a subjectto mount an immune response, the time period for a subject to clearmicroorganism from normal tissue, or the time period for microorganismalproliferation in the tumor or metastasis. In one example, the timeperiod can be a function of the time period for a subject to mount animmune response; for example, the time period can be more than the timeperiod for a subject to mount an immune response, such as more thanabout one week, more than about ten days, more than about two weeks, ormore than about a month; in another example, the time period can be lessthan the time period for a subject to mount an immune response, such asless than about one week, less than about ten days, less than about twoweeks, or less than about a month. In another example, the time periodcan be a function of the time period for a subject to clearmicroorganism from normal tissue; for example, the time period can bemore than the time period for a subject to clear microorganism fromnormal tissue, such as more than about a day, more than about two days,more than about three days, more than about five days, or more thanabout a week. In another example, the time period can be a function ofthe time period for microorganismal proliferation in the tumor ormetastasis; for example, the time period can be more than the amount oftime for a detectable signal to arise in a tumor or metastasis afteradministration of a microorganism expressing a detectable marker, suchas about 3 days, about 5 days, about a week, about ten days, about twoweeks, or about a month.

e. Co-Administrations

Also provided are methods in which an additional therapeutic substance,such as a different therapeutic microorganism or a therapeutic compoundis administered. These can be administered simultaneously, sequentiallyor intermittently with the first microorganism. The additionaltherapeutic substance can interact with the microorganism or a geneproduct thereof, or the additional therapeutic substance can actindependently of the microorganism.

i. Administration of a Plurality of Microorganisms

Methods are provided for administering to a subject two or moremicroorganisms. Administration can be effected simultaneously,sequentially or intermittently. The plurality of microorganisms can beadministered as a single composition or as two or more compositions. Thetwo or more microorganisms can include at least two bacteria, at leasttwo viruses, at least two eukaryotic cells, or two or more selected fromamong bacteria, viruses and eukaryotic cells. The plurality ofmicroorganisms can be provided as combinations of compositionscontaining and/or as kits that include the microorganisms packaged foradministration and optionally including instructions therefore. Thecompositions can contain the microorganisms formulated for single dosageadministration (i.e., for direct administration) and can requiredilution or other additions.

In one embodiment, at least one of the microorganisms is a modifiedmicroorganism such as those provided herein, having a characteristicsuch as low pathogenicity, low toxicity, preferential accumulation intumor, ability to activate an immune response against tumor cells,immunogenic, replication competent, ability to express exogenousproteins, and combinations thereof. The microorganisms can beadministered at approximately the same time, or can be administered atdifferent times. The microorganisms can be administered in the samecomposition or in the same administration method, or can be administeredin separate composition or by different administration methods.

In one example, a bacteria and a virus can be administered to a subject.The bacteria and virus can be administered at the same time, or atdifferent times. For example, the virus can be administered prior toadministering the bacteria, or the bacteria can be administered prior toadministering the virus; typically the virus is administered prior toadministering the bacteria. As provided herein, administering to asubject a virus prior to administering to the subject a bacterium canincrease the amount of bacteria that can accumulate and/or proliferatein a tumor, relative to methods in which bacteria alone areadministered.

Accordingly, the methods provided herein that include administration ofvirus prior to administration of bacteria permit the administration of alower dosage amount of bacteria than would otherwise be administered ina method in which bacteria alone are administered or a method in whichbacteria are administered at the same time as or prior to administrationof a virus. For example, in some embodiments, a bacterium to beadministered can have one or more properties that limit the ability ofthe bacterium to be used, such properties can include, but are notlimited to toxicity, low tumor specificity of accumulation, and limitedproliferation capacity. A bacterium to be administered that has one ormore limiting properties can require administration in lower dosageamounts, or can require assistance in tumor-specific accumulation and/orproliferation. Provided herein are methods of administering such abacterium with limiting properties, where prior to administering thebacterium, a virus is administered such that the limited bacterium canbe administered in smaller quantities, can accumulate in tumor withincreased specificity, and/or can have an increased ability toproliferate in a tumor.

The time period between administrations can be any time period thatachieves the desired effects, as can be determined by one skilled in theart. Selection of a time period between administrations of differentmicroorganisms can be determined according to parameters similar tothose for selecting the time period between administrations of the samemicroorganism, including results from monitoring steps, the time periodfor a subject to mount an immune response, the time period for a subjectto clear microorganism from normal tissue, or the time period formicroorganismal proliferation in the tumor or metastasis. In oneexample, the time period can be a function of the time period for asubject to mount an immune response; for example, the time period can bemore than the time period for a subject to mount an immune response,such as more than about one week, more than about ten days, more thanabout two weeks, or more than about a month; in another example, thetime period can be less than the time period for a subject to mount animmune response, such as less than about one week, less than about tendays, less than about two weeks, or less than about a month. In anotherexample, the time period can be a function of the time period for asubject to clear microorganism from normal tissue; for example, the timeperiod can be more than the time period for a subject to clearmicroorganism from normal tissue, such as more than about a day, morethan about two days, more than about three days, more than about fivedays, or more than about a week. In another example, the time period canbe a function of the time period for microorganismal proliferation inthe tumor or metastasis; for example, the time period can be more thanthe amount of time for a detectable signal to arise in a tumor ormetastasis after administration of a microorganism expressing adetectable marker, such as about 3 days, about 5 days, about a week,about ten days, about two weeks, or about a month. In one example avirus can first be administered, and a bacteria can be administeredabout 5 days after administration of the virus. In another example, avirus can be first administered, and a bacterium can be administeredupon detection of a virally-encoded detectable gene product in the tumorof the subject, optionally when the virally-encoded detectable geneproduct is detected only in the tumor of the subject.

ii. Therapeutic Compounds

The methods can include administering one or more therapeutic compoundsto the subject in addition to administering a microorganism or pluralitythereof to a subject. Therapeutic compounds can act independently, or inconjunction with the microorganism, for tumor therapeutic effects.Therapeutic compounds that can act independently include any of avariety of known chemotherapeutic compounds that can inhibit tumorgrowth, inhibit metastasis growth and/or formation, decrease the size ofa tumor or metastasis, eliminate a tumor or metastasis, without reducingthe ability of a microorganism to accumulate in a tumor, replicate inthe tumor, and cause or enhance an anti-tumor immune response in thesubject.

Therapeutic compounds that act in conjunction with the microorganismsinclude, for example, compounds that alter the expression of themicroorganism or compounds that can interact with amicroorganism-expressed gene, or compounds that can inhibitmicroorganismal proliferation, including compounds toxic to themicroorganism. Therapeutic compounds that can act in conjunction withthe microorganism include, for example, therapeutic compounds thatincrease the proliferation, toxicity, tumor cell killing, or immuneresponse eliciting properties of a microorganism, and also can include,for example, therapeutic compounds that decrease the proliferation,toxicity, or cell killing properties of a microorganism. Thus, providedherein are methods of administering to a subject one or more therapeuticcompounds that can act in conjunction with the microorganism to increasethe proliferation, toxicity, tumor cell killing, or immune responseeliciting properties of a microorganism. Also provided herein aremethods of administering to a subject one or more therapeutic compoundsthat can act in conjunction with the microorganism to decrease theproliferation, toxicity, or cell killing properties of a microorganism.

In one embodiment, therapeutic compounds that can act in conjunctionwith the microorganism to increase the proliferation, toxicity, tumorcell killing, or immune response eliciting properties of a microorganismare compounds that can alter gene expression, where the altered geneexpression can result in an increased killing of tumor cells or anincreased anti-tumor immune response in the subject. A geneexpression-altering compound can, for example, cause an increase ordecrease in expression of one or more microorganismal genes, includingendogenous microorganismal genes and/or exogenous microorganismal genes.For example, a gene expression-altering compound can induce or increasetranscription of a gene in a microorganism such as an exogenous genethat can cause cell lysis or cell death, that can provoke an immuneresponse, that can catalyze conversion of a prodrug-like compound, orthat can inhibit expression of a tumor cell gene. Any of a wide varietyof compounds that can alter gene expression are known in the art,including IPTG and RU486. Exemplary genes whose expression can beup-regulated include proteins and RNA molecules, including toxins,enzymes that can convert a prodrug to an anti-tumor drug, cytokines,transcription regulating proteins, siRNA, and ribozymes. In anotherexample, a gene expression-altering compound can inhibit or decreasetranscription of a gene in a microorganism such as an exogenous genethat can reduce microorganismal toxicity or reduces microorganismalproliferation. Any of a variety of compounds that can reduce or inhibitgene expression can be used in the methods provided herein, includingsiRNA compounds, transcriptional inhibitors or inhibitors oftranscriptional activators. Exemplary genes whose expression can bedown-regulated include proteins and RNA molecules, includingmicroorganismal proteins or RNA that suppress lysis, nucleotidesynthesis or proliferation, and cellular proteins or RNA molecules thatsuppress cell death, immunoreactivity, lysis, or microorganismalreplication.

In another embodiment, therapeutic compounds that can act in conjunctionwith the microorganism to increase the proliferation, toxicity, tumorcell killing, or immune response eliciting properties of a microorganismare compounds that can interact with a microorganism-expressed geneproduct, and such interaction can result in an increased killing oftumor cells or an increased anti-tumor immune response in the subject. Atherapeutic compound that can interact with a microorganism-expressedgene product can include, for example a prodrug or other compound thathas little or no toxicity or other biological activity in itssubject-administered form, but after interaction with amicroorganism-expressed gene product, the compound can develop aproperty that results in tumor cell death, including but not limited to,cytotoxicity, ability to induce apoptosis, or ability to trigger animmune response. A variety of prodrug-like substances are known in theart and an exemplary set of such compounds are disclosed elsewhereherein, where such compounds can include gancyclovir, 5-fluorouracil,6-methylpurine deoxyriboside, cephalosporin-doxorubicin,4-[(2-chloroethyl)(2-mesuloxyethyl)amino]benzoyl-L-glutamic acid,acetominophen, indole-3-acetic acid, CB1954,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin,bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28,1-chloromethyl-5-hydroxy-1,2-dihyro-3H-benz[e]indole,epirubicin-glucuronide, 5′-deoxy-5-fluorouridine, cytosine arabinoside,and linamarin.

In another embodiment, therapeutic compounds that can act in conjunctionwith the microorganism to decrease the proliferation, toxicity, or cellkilling properties of a microorganism are compounds that can inhibitmicroorganismal replication, inhibit microorganismal toxins, or causemicroorganismal death. A therapeutic compound that can inhibitmicroorganismal replication, inhibit microorganismal toxins, or causemicroorganismal death can generally include a compound that can blockone or more steps in the microorganismal life cycle, including, but notlimited to, compounds that can inhibit microorganismal DNA replication,microorganismal RNA transcription, viral coat protein assembly, outermembrane or polysaccharide assembly. Any of a variety of compounds thatcan block one or more steps in a microorganismal life cycle are known inthe art, including any known antibiotic, microorganismal DNA polymeraseinhibitors, microorganismal RNA polymerase inhibitors, inhibitors ofproteins that regulate microorganismal DNA replication or RNAtranscription. In one example, when a microorganism is a bacteria, acompound can be an antibiotic. In another example, a microorganism cancontain a gene encoding a microorganismal life cycle protein, such asDNA polymerase or RNA polymerase that can be inhibited by a compoundthat is, optionally, non-toxic to the host organism.

f. State of Subject

In another embodiment, the methods provided herein for administering amicroorganism to a subject can be performed on a subject in any of avariety of states, including an anesthetized subject, an alert subject,a subject with elevated body temperature, a subject with reduced bodytemperature, or other state of the subject that is known to affect theaccumulation of microorganism in the tumor. As provided herein, it hasbeen determined that a subject that is anesthetized can have a decreasedrate of accumulation of a microorganism in a tumor relative to a subjectthat is not anesthetized. Further provided herein, it has beendetermined that a subject with decreased body temperature can have adecreased rate of accumulation of a microorganism in a tumor relative toa subject with a normal body temperature. Accordingly, provided hereinare methods of administering a microorganism to a subject, where themethods can include administering a microorganism to a subject where thesubject is not under anesthesia, such as general anesthesia; forexample, the subject can be under local anesthesia, or can beunanesthetized. Also provided herein are methods of administering amicroorganism to a subject, where the methods can include administeringa microorganism to a subject with altered body temperature, where thealteration of the body temperature can influence the ability of themicroorganism to accumulate in a tumor; typically, a decrease in bodytemperature can decrease the ability of a microorganism to accumulate ina tumor. Thus, in one exemplary embodiment, a method is provided foradministering a microorganism to a subject, where the method includeselevating the body temperature of the subject to a temperature abovenormal, and administering a microorganism to the subject, where themicroorganism can accumulate in the tumor more readily in the subjectwith higher body temperature relative to the ability of themicroorganism to accumulate in a tumor of a subject with a normal bodytemperature.

2. Monitoring

The methods provided herein can further include one or more steps ofmonitoring the subject, monitoring the tumor, and/or monitoring themicroorganism administered to the subject. Any of a variety ofmonitoring steps can be included in the methods provided herein,including, but not limited to, monitoring tumor size, monitoringanti-(tumor antigen) antibody titer, monitoring the presence and/or sizeof metastases, monitoring the subject's lymph nodes, monitoring thesubject's weight or other health indicators including blood or urinemarkers, monitoring anti-(microorganismal antigen) antibody titer,monitoring microorganismal expression of a detectable gene product, anddirectly monitoring microorganismal titer in a tumor, tissue or organ ofa subject.

The purpose of the monitoring can be simply for assessing the healthstate of the subject or the progress of therapeutic treatment of thesubject, or can be for determining whether or not further administrationof the same or a different microorganism is warranted, or fordetermining when or whether or not to administer a compound to thesubject where the compound can act to increase the efficacy of thetherapeutic method, or the compound can act to decrease thepathogenicity of the microorganism administered to the subject.

a. Monitoring Microorganismal Gene Expression

In some embodiments, the methods provided herein can include monitoringone or more microorganismally expressed genes. Microorganisms, such asthose provided herein or otherwise known in the art, can express one ormore detectable gene products, including but not limited to, detectableproteins.

As provided herein, measurement of a detectable gene product expressedin a microorganism can provide an accurate determination of the level ofmicroorganism present in the subject. As further provided herein,measurement of the location of the detectable gene product, for example,by imaging methods including tomographic methods, can determine thelocalization of the microorganism in the subject. Accordingly, themethods provided herein that include monitoring a detectablemicroorganismal gene product can be used to determine the presence orabsence of the microorganism in one or more organs or tissues of asubject, and/or the presence or absence of the microorganism in a tumoror metastases of a subject. Further, the methods provided herein thatinclude monitoring a detectable microorganismal gene product can be usedto determine the titer of microorganism present in one or more organs,tissues, tumors or metastases. Methods that include monitoring thelocalization and/or titer of microorganisms in a subject can be used fordetermining the pathogenicity of a microorganism; since microorganismalinfection, and particularly the level of infection, of normal tissuesand organs can indicate the pathogenicity of the probe, methods ofmonitoring the localization and/or amount of microorganisms in a subjectcan be used to determine the pathogenicity of a microorganism. Sincemethods provided herein can be used to monitor the amount ofmicroorganisms at any particular location in a subject, the methods thatinclude monitoring the localization and/or titer of microorganisms in asubject can be performed at multiple time points, and, accordingly candetermine the rate of microorganismal replication in a subject,including the rate of microorganismal replication in one or more organsor tissues of a subject; accordingly, the methods of monitoring amicroorganismal gene product can be used for determining the replicationcompetence of a microorganism. The methods provided herein also can beused to quantitate the amount of microorganism present in a variety oforgans or tissues, and tumors or metastases, and can thereby indicatethe degree of preferential accumulation of the microorganism in asubject; accordingly, the microorganismal gene product monitoringmethods provided herein can be used in methods of determining theability of a microorganism to accumulate in tumor or metastases inpreference to normal tissues or organs. Since the microorganisms used inthe methods provided herein can accumulate in an entire tumor or canaccumulate at multiple sites in a tumor, and can also accumulate inmetastases, the methods provided herein for monitoring a microorganismalgene product can be used to determine the size of a tumor or the numberof metastases are present in a subject. Monitoring such presence ofmicroorganismal gene product in tumor or metastasis over a range of timecan be used to assess changes in the tumor or metastasis, includinggrowth or shrinking of a tumor, or development of new metastases ordisappearance of metastases, and also can be used to determine the rateof growth or shrinking of a tumor, or development of new metastases ordisappearance of metastases, or the change in the rate of growth orshrinking of a tumor, or development of new metastases or disappearanceof metastases. Accordingly, the methods of monitoring a microorganismalgene product can be used for monitoring a neoplastic disease in asubject, or for determining the efficacy of treatment of a neoplasticdisease, by determining rate of growth or shrinking of a tumor, ordevelopment of new metastases or disappearance of metastases, or thechange in the rate of growth or shrinking of a tumor, or development ofnew metastases or disappearance of metastases.

Any of a variety of detectable proteins can be detected in themonitoring methods provided herein; an exemplary, non-limiting list ofsuch detectable proteins includes any of a variety of fluorescenceproteins (e.g., green fluorescence proteins), any of a variety ofluciferases, transferrin or other iron binding proteins; or receptors,binding proteins, and antibodies, where a compound that specificallybinds the receptor, binding protein or antibody can be a detectableagent or can be labeled with a detectable substance (e.g., aradionuclide or imaging agent).

b. Monitoring Tumor Size

Also provided herein are methods of monitoring tumor and/or metastasissize and location. Tumor and/or metastasis size can be monitored by anyof a variety of methods known in the art, including external assessmentmethods or tomographic or magnetic imaging methods. In addition to themethods known in the art, methods provided herein, for example,monitoring microorganismal gene expression, can be used for monitoringtumor and/or metastasis size.

Monitoring size over several time points can provide informationregarding the increase or decrease in size of a tumor or metastasis, andcan also provide information regarding the presence of additional tumorsand/or metastases in the subject. Monitoring tumor size over severaltime points can provide information regarding the development of aneoplastic disease in a subject, including the efficacy of treatment ofa neoplastic disease in a subject.

c. Monitoring Antibody Titer

The methods provided herein also can include monitoring the antibodytiter in a subject, including antibodies produced in response toadministration of a microorganism to a subject. The microorganismsadministered in the methods provided herein can elicit an immuneresponse to endogenous microorganismal antigens. The microorganismsadministered in the methods provided herein also can elicit an immuneresponse to exogenous genes expressed by a microorganism. Themicroorganisms administered in the methods provided herein also canelicit an immune response to tumor antigens. Monitoring antibody titeragainst microorganismal antigens, microorganismally expressed exogenousgene products, or tumor antigens can be used in methods of monitoringthe toxicity of a microorganism, monitoring the efficacy of treatmentmethods, or monitoring the level of gene product or antibodies forproduction and/or harvesting.

In one embodiment, monitoring antibody titer can be used to monitor thetoxicity of a microorganism. Antibody titer against a microorganism canvary over the time period after administration of the microorganism tothe subject, where at some particular time points, a lowanti-(microorganismal antigen) antibody titer can indicate a highertoxicity, while at other time points a high anti-(microorganismalantigen) antibody titer can indicate a higher toxicity. Themicroorganisms used in the methods provided herein can be immunogenic,and can, therefore, elicit an immune response soon after administeringthe microorganism to the subject. Generally, a microorganism againstwhich a subject's immune system can quickly mount a strong immuneresponse can be a microorganism that has low toxicity when the subject'simmune system can remove the microorganism from all normal organs ortissues. Thus, in some embodiments, a high antibody titer againstmicroorganismal antigens soon after administering the microorganism to asubject can indicate low toxicity of a microorganism. In contrast, amicroorganism that is not highly immunogenic may infect a host organismwithout eliciting a strong immune response, which can result in a highertoxicity of the microorganism to the host. Accordingly, in someembodiments, a high antibody titer against microorganismal antigens soonafter administering the microorganism to a subject can indicate lowtoxicity of a microorganism.

In other embodiments, monitoring antibody titer can be used to monitorthe efficacy of treatment methods. In the methods provided herein,antibody titer, such as anti-(tumor antigen) antibody titer, canindicate the efficacy of a therapeutic method such as a therapeuticmethod to treat neoplastic disease. Therapeutic methods provided hereincan include causing or enhancing an immune response against a tumorand/or metastasis. Thus, by monitoring the anti-(tumor antigen) antibodytiter, it is possible to monitor the efficacy of a therapeutic method incausing or enhancing an immune response against a tumor and/ormetastasis. The therapeutic methods provided herein also can includeadministering to a subject a microorganism that can accumulate in atumor and can cause or enhance an anti-tumor immune response.Accordingly, it is possible to monitor the ability of a host to mount animmune response against microorganisms accumulated in a tumor ormetastasis, which can indicate that a subject has also mounted ananti-tumor immune response, or can indicate that a subject is likely tomount an anti-tumor immune response, or can indicate that a subject iscapable of mounting an anti-tumor immune response.

In other embodiments, monitoring antibody titer can be used formonitoring the level of gene product or antibodies for production and/orharvesting. As provided herein, methods can be used for producingproteins, RNA molecules or other compounds by expressing an exogenousgene in a microorganism that has accumulated in a tumor. Furtherprovided herein are methods for producing antibodies against a protein,RNA molecule or other compound produced by exogenous gene expression ofa microorganism that has accumulated in a tumor. Monitoring antibodytiter against the protein, RNA molecule or other compound can indicatethe level of production of the protein, RNA molecule or other compoundby the tumor-accumulated microorganism, and also can directly indicatethe level of antibodies specific for such a protein, RNA molecule orother compound.

d. Monitoring General Health Diagnostics

The methods provided herein also can include methods of monitoring thehealth of a subject. Some of the methods provided herein are therapeuticmethods, including neoplastic disease therapeutic methods. Monitoringthe health of a subject can be used to determine the efficacy of thetherapeutic method, as is known in the art. The methods provided hereinalso can include a step of administering to a subject a microorganism.Monitoring the health of a subject can be used to determine thepathogenicity of a microorganism administered to a subject. Any of avariety of health diagnostic methods for monitoring disease such asneoplastic disease, infectious disease, or immune-related disease can bemonitored, as is known in the art. For example, the weight, bloodpressure, pulse, breathing, color, temperature or other observable stateof a subject can indicate the health of a subject. In addition, thepresence or absence or level of one or more components in a sample froma subject can indicate the health of a subject. Typical samples caninclude blood and urine samples, where the presence or absence or levelof one or more components can be determined by performing, for example,a blood panel or a urine panel diagnostic test. Exemplary componentsindicative of a subject's health include, but are not limited to, whiteblood cell count, hematocrit, c-reactive protein concentration.

e. Monitoring Coordinated with Treatment

Also provided herein are methods of monitoring a therapy, wheretherapeutic decisions can be based on the results of the monitoring.Therapeutic methods provided herein can include administering to asubject a microorganism, where the microorganism can preferentiallyaccumulate in a tumor and/or metastasis, and where the microorganism cancause or enhance an anti-tumor immune response. Such therapeutic methodscan include a variety of steps including multiple administrations of aparticular microorganism, administration of a second microorganism, oradministration of a therapeutic compound. Determination of the amount,timing or type of microorganism or compound to administer to the subjectcan be based on one or more results from monitoring the subject. Forexample, the antibody titer in a subject can be used to determinewhether or not it is desirable to administer a microorganism orcompound, the quantity of microorganism or compound to administer, andthe type of microorganism or compound to administer, where, for example,a low antibody titer can indicate the desirability of administeringadditional microorganism, a different microorganism, or a therapeuticcompound such as a compound that induces microorganismal geneexpression. In another example, the overall health state of a subjectcan be used to determine whether or not it is desirable to administer amicroorganism or compound, the quantity of microorganism or compound toadminister, and the type of microorganism or compound to administer,where, for example, determining that the subject is healthy can indicatethe desirability of administering additional microorganism, a differentmicroorganism, or a therapeutic compound such as a compound that inducesmicroorganismal gene expression. In another example, monitoring adetectable microorganismally expressed gene product can be used todetermine whether or not it is desirable to administer a microorganismor compound, the quantity of microorganism or compound to administer,and the type of microorganism or compound to administer. Such monitoringmethods can be used to determine whether or not the therapeutic methodis effective, whether or not the therapeutic method is pathogenic to thesubject, whether or not the microorganism has accumulated in a tumor ormetastasis, and whether or not the microorganism has accumulated innormal tissues or organs. Based on such determinations, the desirabilityand form of further therapeutic methods can be derived.

In one embodiment, determination of whether or not a therapeutic methodis effective can be used to derive further therapeutic methods. Any of avariety of methods of monitoring can be used to determine whether or nota therapeutic method is effective, as provided herein or otherwise knownin the art. If monitoring methods indicate that the therapeutic methodis effective, a decision can be made to maintain the current course oftherapy, which can include further administrations of a microorganism orcompound, or a decision can be made that no further administrations arerequired. If monitoring methods indicate that the therapeutic method isineffective, the monitoring results can indicate whether or not a courseof treatment should be discontinued (e.g., when a microorganism ispathogenic to the subject), or changed (e.g., when a microorganismaccumulates in a tumor without harming the host organism, but withouteliciting an anti-tumor immune response), or increased in frequency oramount (e.g., when little or no microorganism accumulates in tumor).

In one example, monitoring can indicate that a microorganism ispathogenic to a subject. In such instances, a decision can be made toterminate administration of the microorganism to the subject, toadminister lower levels of the microorganism to the subject, toadminister a different microorganism to a subject, or to administer to asubject a compound that reduces the pathogenicity of the microorganism.In one example, administration of a microorganism that is determined tobe pathogenic can be terminated. In another example, the dosage amountof a microorganism that is determined to be pathogenic can be decreasedfor subsequent administration; in one version of such an example, thesubject can be pre-treated with another microorganism that can increasethe ability of the pathogenic microorganism to accumulate in tumor,prior to re-administering the pathogenic microorganism to the subject.In another example, a subject can have administered thereto a bacteriaor virus that is pathogenic to the subject; administration of such apathogenic microorganism can be accompanied by administration of, forexample an antibiotic, anti-microorganismal compound, pathogenicityattenuating compound (e.g., a compound that down-regulates theexpression of a lytic or apoptotic gene product), or other compound thatcan decrease the proliferation, toxicity, or cell killing properties ofa microorganism, as described herein elsewhere. In one variation of suchan example, the localization of the microorganism can be monitored, and,upon determination that the microorganism is accumulated in tumor and/ormetastases but not in normal tissues or organs, administration of theantibiotic, anti-microorganismal compound or pathogenicity attenuatingcompound can be terminated, and the pathogenic activity of themicroorganism can be activated or increased, but limited to the tumorand/or metastasis. In another variation of such an example, afterterminating administration of an antibiotic, anti-microorganismalcompound or pathogenicity attenuating compound, the presence of themicroorganism and/or pathogenicity of the microorganism can be furthermonitored, and administration of such a compound can be reinitiated ifthe microorganism is determined to pose a threat to the host by, forexample, spreading to normal organs or tissues, releasing a toxin intothe vasculature, or otherwise having pathogenic effects reaching beyondthe tumor or metastasis.

In another example, monitoring can determine whether or not amicroorganism has accumulated in a tumor or metastasis of a subject.Upon such a determination, a decision can be made to further administeradditional microorganism, a different microorganism or a compound to thesubject. In one example, monitoring the presence of a virus in a tumoror metastasis can be used in deciding to administer to the subject abacterium, where, for example, the quantity of bacteria administered canbe reduced according to the presence and/or quantity of virus in a tumoror metastasis. In a similar example, monitoring the presence of a virusin a tumor or metastasis can be used in deciding when to administer tothe subject a bacterium, where, for example, the bacteria can beadministered upon detecting to the presence and/or a selected quantityof virus in a tumor or metastasis. In another example, monitoring thepresence of a microorganism in a tumor can be used in deciding toadminister to the subject a compound, where the compound can increasethe pathogenicity, proliferation, or immunogenicity of a microorganismor the compound can otherwise act in conjunction with the microorganismto increase the proliferation, toxicity, tumor cell killing, or immuneresponse eliciting properties of a microorganism; in one variation ofsuch an example, the microorganism can, for example have little or nolytic or cell killing capability in the absence of such a compound; in afurther variation of such an example, monitoring of the presence of themicroorganism in a tumor or metastasis can be coupled with monitoringthe absence of the microorganism in normal tissues or organs, where thecompound is administered if the microorganism is present in tumor ormetastasis and not at all present or substantially not present in normalorgans or tissues; in a further variation of such an example, the amountof microorganism in a tumor or metastasis can be monitored, where thecompound is administered if the microorganism is present in tumor ormetastasis at sufficient levels.

E. METHODS OF PRODUCING GENE PRODUCTS AND ANTIBODIES

Provided herein are microorganisms, and methods for making and usingsuch microorganisms for production products of exogenous genes and/orfor production of antibodies specific for exogenous gene products. Themethods provided herein result in efficient recombinant production ofbiologically active proteins. In EP A1 1 281 772, it is disclosed thatwhen vaccinia virus (LIVP strain) carrying the light emitting fusiongene construct rVV-ruc-gfp (RVGL9) was injected intravenously into nudemice, the virus particles were found to be cleared from all internalorgans within 4 days, as determined by extinction of light emission. Incontrast, when the fate of the injected vaccinia virus was similarlyfollowed in nude mice bearing tumors grown from subcutaneously implantedC6 rat glioma cells, virus particles were found to be retained over timein the tumor tissues, resulting in lasting light emission. The presenceand amplification of the virus-encoded fusion proteins in the same tumorwere monitored in live animals by observing GFP fluorescence under astereomicroscope and by detecting luciferase-catalyzed light emissionunder a low-light video-imaging camera. Tumor-specific light emissionwas detected 4 days after viral injection in nude mice carryingsubcutaneous C6 glioma implants. Tumor accumulation of rVV-ruc-gfp(RVGL9) virus particles was also seen in nude mice carrying subcutaneoustumors developed from implanted PC-3 human prostate cells, and in micewith orthotopically implanted MCF-7 human breast tumors. Further,intracranial C6 rat glioma cell implants in immunocompetent rats andMB-49 human bladder tumor cell implants in C57 mice were also targetedby the vaccinia virus. In addition to primary breast tumors, smallmetastatic tumors were also detected externally in the contralateralbreast region, as well as in nodules on the exposed lung surface,suggesting metastasis to the contralateral breast and lung. In summaryit was shown that light-emitting cells or microorganisms, for example,vaccinia virus can be used to detect and treat metastatic tumors.

Similar results were obtained with light-emitting bacteria (Salmonella,Vibrio, Listeria, E. coli) which were injected intravenously into miceand which could be visualized in whole animals under a low light imagerimmediately. No light emission was detected twenty four hours afterbacterial injection in both athymic (nu/nu) mice and immunocompetent C57mice as a result of clearing by the immune system. In nude mice bearingtumors developed from implanted C6 glioma cells, light emission wasabolished from the animal entirely twenty four hours after delivery ofbacteria, similar to mice without tumors. However, forty eight hourspost-injection, a strong, rapidly increasing light emission originatedonly from the tumor regions was observed. This observation indicated acontinuous bacterial replication in the tumor tissue. The extent oflight emission was dependent on the bacterial strain used. The homing-inprocess together with the sustained light emission was also demonstratedin nude mice carrying prostate, bladder, and breast tumors. In additionto primary tumors, metastatic tumors could also be visualized asexemplified in the breast tumor model. Tumor-specific light emission wasalso observed in immunocompetent C57 mice, with bladder tumors as wellas in Lewis rats with brain glioma implants. Once in the tumor, thelight-emitting bacteria were not observed to be released into thecirculation and to re-colonize subsequently implanted tumors in the sameanimal. Further, mammalian cells expressing the Ruc-GFP fusion protein,upon injection into the bloodstream, were also found to home in to, andpropagate in, glioma tumors. These findings opened the way for designingmultifunctional viral vectors useful for the detection of tumors basedon signals such as light emission, for suppression of tumor developmentand angiogenesis signaled by, for example, light extinction and thedevelopment of bacterial and mammalian cell-based tumor targetingsystems in combination with therapeutic gene constructs for thetreatment of cancer. These systems have the following advantages: (a)They target the tumor specifically without affecting normal tissue; (b)the expression and secretion of the therapeutic gene constructs can be,optionally, under the control of an inducible promoter enablingsecretion to be switched on or off; and (c) the location of the deliverysystem inside the tumor can be verified by direct visualization beforeactivating gene expression and protein delivery.

As provided herein, the system described above based on the accumulationof bacteria, viruses and eukaryotic cells in tumors can be used forsimple, quick, and inexpensive production of proteins and otherbiological compounds originating from cloned nucleotide sequences. Thissystem also is useful for the concomitant overproduction ofpolypeptides, RNA or other biological compounds (in tumor tissue) andantibodies against those compounds (in the serum) in the same animal. Asprovided herein, after intravenous injection, a microorganism such asvaccinia virus can enter the tumor of an animal and, due to theimmunoprivileged state of the tumor, can replicate preferentially in thetumor tissues and thereby can overproduce the inserted gene encodedprotein in the tumors. After harvesting the tumor tissues, the localizedand overexpressed protein can be isolated by a simple procedure fromtumor homogenates. In addition, based on the findings that only 0.2 to0.3% of the desired proteins produced in the tumor were found in theblood stream of the same animal, a simultaneous vaccination of the mouseand efficient antibody production against the overproduced protein wasachieved. Thus, serum from the same mouse (or any other animal) can beharvested and used as mouse-derived antibodies against the proteins orother products overproduced in the tumor.

Thus, provided herein are methods of producing gene products and orantibodies in a non-human subject, by administering to a subjectcontaining a tumor, a microorganism, where the microorganism expresses aselected protein or RNA to be produced, a protein or RNA whoseexpression can result in the formation of a compound to be produced, ora selected protein or RNA against which an antibody is to be produced.The methods provided herein can further include administering to asubject containing a tumor, a microorganism expressing an exogenous geneencoding a selected protein or RNA to be produced, a protein or RNAwhose expression can result in the formation of a compound to beproduced, or a selected protein or RNA against which an antibody is tobe produced. The methods provided herein can further includeadministering to a subject containing a tumor, a microorganismexpressing a gene encoding a selected protein or RNA to be produced, aprotein or RNA whose expression can result in the formation of acompound to be produced, or a selected protein or RNA against which anantibody is to be produced, where such gene expression can be regulated,for example, by a transcriptional activator or inducer, or atranscriptional suppressor. The methods provided herein for producing aprotein, RNA, compound or antibody can further include monitoring thelocalization and/or level of the microorganism in the subject bydetecting a detectable protein, where the detectable protein canindicate the expression of the selected gene, or can indicate thereadiness of the microorganism to be induced to express the selectedgene or for suppression of expression to be terminated or suspended.Also provided herein are methods of producing gene products and orantibodies in a non-human subject, by administering to a subjectcontaining a tumor, a microorganism, where the microorganism expresses aselected protein or RNA to be produced, a protein or RNA whoseexpression can result in the formation of a compound to be produced, ora selected protein or RNA against which an antibody is to be produced,where the subject to which the microorganism is administered is not atransgenic animal. Also provided herein are methods of producing geneproducts and or antibodies in a non-human subject, by administering to asubject containing a tumor, a microorganism, where the microorganismexpresses a selected protein to be produced, where the tumor within thesubject is selected according to its ability to post-translationallyprocess the selected protein.

The advantages of the system, include:

(a) No production of a transgenic animal carrying the novelpolypeptide-encoding cassette is required;(b) the tumor system is more efficient than tissue culture;(c) proteins interfering with animal development and other toxicproteins can be overproduced in tumors without negative effects to thehost animal;(d) the system is fast: within 4 to 6 weeks from cDNA cloning to proteinand antisera purification;(e) the system is relatively inexpensive and can be scaled up easily;(f) correct protein folding and modifications can be achieved;(g) high antigenicity can be achieved, which is beneficial for betterantibody production; and(h) species-specific-cell-based production of proteins in animals suchas mice, with tumors as fermentors can be achieved.

Depiction of an exemplary method for production of gene products and/orantibodies against gene products is provided in FIG. 2.

In one embodiment, methods are provided for producing a desiredpolypeptide, RNA or compound, the method including the following steps:(a) injecting a microorganism containing a nucleotide sequence encodingthe desired polypeptide or RNA into an animal bearing a tumor; (b)harvesting the tumor tissue from the animal; and (c) isolating thedesired polypeptide, RNA or compound from the tumor tissue.

Steps of an exemplary method can be summarized as follows (shown for aparticular embodiment, i.e. vaccinia virus additionally containing agene encoding a light-emitting protein):

(1) Insertion of the desired DNA or cDNA into the vaccinia virus genome;(2) modification of the vaccinia virus genome with light-emittingprotein construct as expression marker;(3) recombination and virus assembly in cell culture;(4) screening of individual viral particles carrying inserts followed bylarge scale virus production and concentration;(5) administration of the viral particles into mice or other animalsbearing tumors of human, non-human primate or other mammalian origins;(6) verification of viral replication and protein overproduction inanimals based on light emission;(7) harvest of tumor tissues and, optionally, the blood (separately);and(8) purification of overexpressed proteins from tumors and, optionally,antisera from blood using conventional methods.

Any microorganism can be used in the methods provided herein, providedthat they replicate in the animal, are not pathogenic for the animal,for example, are attenuated, and are recognized by the immune system ofthe animal. In some embodiments, such microorganisms also can expressexogenous genes. Suitable microorganisms and cells are, for example,disclosed in EP A1 1 281 772 and EP A1 1 281 767. The person skilled inthe art also knows how to generate animals carrying the desired tumor(see, e.g., EP A1 1 281 767 or EP A1 1 281 777).

Also provided is a method for simultaneously producing a desiredpolypeptide, RNA or compound and an antibody directed to thepolypeptide, RNA or compound, the method having the following steps: (a)administering a microorganism containing a nucleotide sequence encodingthe desired polypeptide or RNA into an animal bearing a tumor; (b)harvesting the tumor tissue from the animal; (c) isolating the desiredpolypeptide, RNA or compound from the tumor tissue; and (d) isolatingthe antibody directed to the polypeptide, RNA or compound from the serumobtained from the animal. This approach can be used for generatingpolypeptides and/or antibodies against the polypeptides which are toxicor unstable, or which require species specific cellular environment forcorrect folding or modifications.

In another embodiment, the microorganism can further contain anucleotide sequence encoding a detectable protein, such as a luminescentor fluorescent protein, or a protein capable of inducing a detectablesignal.

Typically in methods for transfecting the microorganisms or cells withnucleotide sequences encoding the desired polypeptide or RNA and,optionally, a nucleotide sequence encoding a detectable protein such asa luminescent or fluorescent protein, or a protein capable of inducing adetectable signal, the nucleotide sequences are present in a vector oran expression vector. A person skilled in the art is familiar with avariety of expression vectors, which can be selected according to themicroorganism used to infect the tumor, the cell type of the tumor, theorganism to be infected, and other factors known in the art. In someembodiments, the microorganism can be a virus, including the virusesdisclosed herein. Thus, the nucleotide sequences can be contained in arecombinant virus containing appropriate expression cassettes. Suitableviruses for use herein, include, but are not limited to, baculovirus,vaccinia, Sindbis virus, Sendai virus, adenovirus, an AAV virus or aparvovirus, such as MVM or H-1. The vector can also be a retrovirus,such as MoMULV, MoMuLV, HaMuSV, MuMTV, RSV or GaLV. For expression inmammalian cells, a suitable promoter is, for example, humancytomegalovirus immediate early promoter (pCMV). Furthermore, tissueand/or organ specific promoters can be used. For example, the nucleotidesequences can be operatively linked with a promoter allowing highexpression. Such promoters can include, for example, induciblepromoters; a variety of such promoters are known to persons skilled inthe art.

For generating protein or RNA-encoding nucleotide sequences and forconstructing expression vectors or viruses that contain the nucleotidesequences, it is possible to use general methods known in the art. Thesemethods include, for example, in vitro recombination techniques,synthetic methods and in vivo recombination methods as known in the art,and exemplified in Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. Methods of transfecting cells, of phenotypicallyselecting transfectants cells, of phenotypically selecting transfectantsand of expressing the nucleotide sequences by using vectors containingprotein or RNA-encoding DNA are known in the art.

In some embodiments, the protein or RNA to be produced in the tumor canbe linked to an inducible promoter, such as a promoter that can beinduced by a substance endogenous to the subject, or by a substance thatcan be administered to a subject. Accordingly, provided herein aremethods of producing a protein or RNA in a tumor, where the productioncan be induced by administration of a substance to a subject, and,optionally, harvesting the tumor and isolating the protein or RNA fromthe tumor. Such induction methods can be coupled with methods ofmonitoring a microorganism in a subject. For example, a microorganismcan be monitored by detecting a detectable protein. In methods thatinclude monitoring, detection of a desired localization and/or level ofmicroorganism in the subject can be coordinated with induction ofmicroorganismal gene expression. For example, when a microorganismallyexpressed detectable protein is detected in tumor, but not appreciablyin normal organs or tissues, an inducer can be administered to thesubject. In another example, when a microorganismally expresseddetectable protein is detected in tumor, and also in normal organs ortissues, administration of an inducer can be suspended or postponeduntil the detectable protein is no longer detected in normal organs ortissues. In another example, when a microorganismally expresseddetectable protein is detected at sufficient levels in tumor, an inducercan be administered to the subject. In another example, when amicroorganismally expressed detectable protein is not detected atsufficient levels in tumor administration of an inducer can be suspendedor postponed until the detectable protein is detected at sufficientlevels in the tumor.

Also provided herein are methods of producing a protein or RNA in atumor, by administering a microorganism encoding the protein or RNA, anda suppressor of gene expression. The suppressor of gene expression canbe administered for a pre-defined period of time, or until themicroorganism accumulated in tumor but not in normal organs or tissues,or until sufficient levels of the microorganism have accumulated in thetumor, at which point administration of the suppressor can be terminatedor suspended, which can result in expression of the protein or RNA. Aswill be recognized by one skilled in the art, methods similar to thoseprovided herein in regard to monitoring a detectable protein andadministering an inducer, can also apply for terminating or suspendingadministration of a suppressor.

In one embodiment, the microorganism is a bacterium, for example, anattenuated bacterium, such as those provided herein. Exemplary bacteriainclude attenuated Salmonella typhimurium, attenuated Vibrio cholerae,attenuated Listeria monocytogenes or E. coli. Alternatively, virusessuch as vaccinia virus, AAV, a retrovirus can be used in the methodsprovided herein. In exemplary methods, the virus is vaccinia virus.Other cells that can be used in the present methods include mammaliancells, such as fibroma cells, including human cells such as humanfibroma cells.

Any of a variety of animals, including laboratory or livestock animalscan be used, including for example, mice, rats and other rodents,rabbits, guinea pigs, pigs, sheep, goats, cows and horses. Exemplaryanimals are mice. The tumor can be generated by implanting tumor cellsinto the animal. Generally, for the production of a desired polypeptide,RNA, or compound, any solid tumor type can be used, such as a fastgrowing tumor type. Exemplary fast growing tumor types include C6 ratglioma and HCT116 human colon carcinoma. Generally, for the productionof a desired antibody, a relatively slow growing tumor type can be used.Exemplary slow growing tumor types include HT1080 human fibrosarcoma andGI-101A human breast carcinoma. For T-independent antibody production,nu−/nu− mice bearing allogenic tumor or xenografts can be used; whilefor T-dependent antibody production, immunocompetent mice with syngenictumors can be used. In some embodiments, such as where the compound tobe produced is a protein, the microorganism selected can be amicroorganism that uses the translational components (e.g., proteins,vesicles, substrates) of the tumor cells, such as, for example, a virusthat uses the translational components of a tumor cell. In suchinstances, the tumor cell type can be selected according to the desiredpost-translational processing to be performed on the protein, includingproteolysis, glycosylation, lipidylation, disulfide formation, and anyrefolding or multimer assembly that can require cellular components forcompleting. In some examples, the tumor cell type selected can be thesame species as the protein to be expressed, thus resulting inspecies-specific post-translational processing of the protein; anexemplary tumor cell type-expressed protein species is human.

1. Production of Recombinant Proteins and RNA Molecules

The tumor tissue can be surgically removed from the animal. Afterhomogenization of the tumor tissue, the desired polypeptide, RNA orother biological compound can be purified according to establishedmethods. For example, in the case of a recombinant polypeptide, thepolypeptide might contain a bindable tag such as a his-tag, and can bepurified, for example, via column chromatography. The time necessary foraccumulation of sufficient amounts of the polypeptide or RNA in thetumor of the animal depends on many factors, for example, the kind ofanimal or the kind of tumor, and can be determined by the skilled personby routine experimentation. In general, expression of the desiredpolypeptide can be detected two days after virus injection. Theexpression peaks approximately two weeks after injection, and lasts upto two months. In some embodiments, the amount of desired polypeptide orRNA in the tumor can be determined by monitoring a microorganismallyexpressed detectable substance, where the concentration of thedetectable substance can reflect the amount of desired polypeptide orRNA in the tumor.

In another embodiment, the desired polypeptide, RNA or other compoundcan be manufactured in the subject, and provide a beneficial effect tothe subject. In one example, a microorganism can encode a protein orRNA, or a protein that manufactures a compound that is not manufacturedby the subject. In one example, a microorganism can encode a peptidehormone or cytokine, such as insulin, which can be released into thevasculature of a subject lacking the ability to produce insulin orrequiring increased insulin concentrations in the vasculature. Inanother example, blood clotting factors can be manufactured in a subjectwith blood clotting deficiency, such as a hemophiliac. In someembodiments, the protein or RNA to be produced in the tumor can belinked to an inducible promoter, such as a promoter that can be inducedby increased glucose concentrations. In such instances, the manufactureof the protein or RNA can be controlled in response to one or moresubstances in the subject or by one or more substances that can beadministered to a subject, such as a compound that can inducetranscription, for example, RU486. Thus, in some embodiments, themethods provided herein can include administering to a subject having atumor, a microorganism that can express one or more genes encoding abeneficial gene product or a gene product that can manufacture abeneficial compound.

2. Production of Antibodies

Also provided are methods for producing a desired antibody, the methodcomprising the following steps: (a) administering a microorganismcontaining a nucleotide sequence encoding an antigen into an animalbearing a tumor; and (b) isolating the antibody directed to the antigenfrom the serum obtained from the animal. The antibodies directed to theantigen can be isolated and purified according to well known methods.Antibodies that are directed against specific contaminating antigens(e.g., bacteria antigens) can be removed by adsorption, and theantibodies directed against the target antigen can be separated fromcontaminating antibodies by affinity purification, for example, byimmuno affinity chromatography using the recombinant antigen as theligand of the column, by methods known in the art. Antibodies can becollected from the animal in a single harvest, or can be collected overtime by collection bleeds, as is known in the art.

F. PHARMACEUTICAL COMPOSITIONS, COMBINATIONS AND KITS

Provided herein are pharmaceutical compositions, combinations and kitscontaining a microorganism provided herein and one or more components.Pharmaceutical compositions can include a microorganism and apharmaceutical carrier. Combinations can include two or moremicroorganisms, a microorganism and a detectable compound, amicroorganism and a microorganism expression modulating compound, amicroorganism and a therapeutic compound. Kits can include thepharmaceutical compositions and/or combinations provided herein, and oneor more components such as instructions for use, a device for detectinga microorganism in a subject, a device for administering a compound to asubject, and a device for administering a compound to a subject.

1. Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions containing amodified microorganism and a suitable pharmaceutical carrier. Examplesof suitable pharmaceutical carriers are known in the art and includephosphate buffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions, etc. Suchcarriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Colloidal dispersionsystems that can be used for delivery of microorganisms includemacromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions (mixed), micelles,liposomes and lipoplexes. An exemplary colloidal system is a liposome.Organ-specific or cell-specific liposomes can be used in order toachieve delivery only to the desired tissue. The targeting of liposomescan be carried out by the person skilled in the art by applying commonlyknown methods. This targeting includes passive targeting (utilizing thenatural tendency of the liposomes to distribute to cells of the RES inorgans which contain sinusoidal capillaries) or active targeting (forexample by coupling the liposome to a specific ligand, for example, anantibody, a receptor, sugar, glycolipid, protein etc., by well knownmethods). In the present methods, monoclonal antibodies can be used totarget liposomes to specific tissues, for example, tumor tissue, viaspecific cell-surface ligands.

2. Host Cells

Also provided herein are host cells that contain a microorganismprovided herein such as a modified vaccinia virus. These host cells caninclude any of a variety of mammalian, avian and insect cells andtissues that are susceptible to microorganisms, such as vaccinia virusinfection, including chicken embryo, rabbit, hamster and monkey kidneycells, for example, CV-1, BSC40, Vero, BSC40 and BSC-1, and human HeLacells. Methods of transforming these host cells, of phenotypicallyselecting transformants etc., are known in the art.

3. Combinations

Combinations can include a microorganism and one or more components. Anycombination herein also can, in place of a microorganism, contain apharmaceutical composition and/or a host cell containing a microorganismand one or more components.

Exemplary combinations can contain two or more microorganisms, amicroorganism and a detectable compound, a microorganism and amicroorganism expression modulating compound, or a microorganism and atherapeutic compound. Combinations that contain two or moremicroorganisms can contain, for example, two or more microorganisms thatcan both be administered to a subject in performing the methods providedherein, including sequentially administering the tow microorganisms. Inone example, a combination can contain a virus and a bacterium, where,for example, the virus can first be administered to the subject, and thebacterium can be subsequently administered to the subject.

Combinations provided herein can contain a microorganism and adetectable compound. A detectable compound can include a ligand orsubstrate or other compound that can interact with and/or bindspecifically to a microorganismally expressed protein or RNA molecule,and can provide a detectable signal, such as a signal detectable bytomographic, spectroscopic or magnetic resonance techniques. Exemplarydetectable compounds can be, or can contain, an imaging agent such as amagnetic resonance, ultrasound or tomographic imaging agent, including aradionuclide. The detectable compound can include any of a variety ofcompounds as provided elsewhere herein or are otherwise known in theart. Typically, the detectable compound included with a microorganism inthe combinations provided herein will be a compound that is a substrate,a ligand, or can otherwise specifically interact with, a protein or RNAencoded by the microorganism; in some examples, the protein or RNA is anexogenous protein or RNA. Exemplary microorganisms/detectable compoundsinclude a microorganism encoding luciferase/luciferin,β-galactosidase/(4,7,10-tri(aceticacid)-1-(2-β-galactopyranosylethoxy)-1,4,7,10-tetraazacyclododecane)gadolinium (Egad), and other combinations known in the art.

Combinations provided herein can contain a microorganism and amicroorganism gene expression modulating compound. Compounds thatmodulate gene expression are known in the art, and include, but are notlimited to, transcriptional activators, inducers, transcriptionalsuppressors, RNA polymerase inhibitors, and RNA binding compounds suchas siRNA or ribozymes. Any of a variety of gene expression modulatingcompounds known in the art can be included in the combinations providedherein. Typically, the gene expression modulating compound included witha microorganism in the combinations provided herein will be a compoundthat can bind, inhibit, or react with one or more compounds active ingene expression such as a transcription factor or RNA, of themicroorganism of the combination. An exemplary microorganism/expressionmodulator can be a microorganism encoding a chimeric transcriptionfactor complex having a mutant human progesterone receptor fused to ayeast GAL4 DNA-binding domain an activation domain of the herpes simplexvirus protein VP16 and also containing a synthetic promoter containing aseries of GAL4 recognition sequences upstream of the adenovirus majorlate E1B TATA box, where the compound can be RU486 (see, e.g., Yu etal., Mol Genet Genomics 2002 268:169-178). A variety of othermicroorganism/expression modulator combinations known in the art alsocan be included in the combinations provided herein.

Combinations provided herein can contain a microorganism and atherapeutic compound. Therapeutic compounds can include compounds thatare substrates for microorganismally expressed enzymes, compound thatcan kill or inhibit microorganism growth or toxicity, or othertherapeutic compounds provided herein or known in the art to act inconcert with a microorganism. Typically, the therapeutic compoundincluded with a microorganism in the combinations provided herein willbe a compound that can act in concert with a microorganism, such as asubstrate of an enzyme encoded by the microorganism, or anantimicroorganismal agent known to be effective against themicroorganism of the combination. Exemplary microorganism/therapeuticcompound combinations can include a microorganism encoding Herpessimplex virus thymidine kinase/gancyclovir, and streptococcuspyogenes/penicillin. Any of a variety of known combinations providedherein or otherwise known in the art can be included in the combinationsprovided herein.

4. Kits

Kits are packaged in combinations that optionally include other reagentsor devices, or instructions for use. Any kit provided herein also can,in place of a microorganism, contain a pharmaceutical composition, ahost cell containing a microorganism, and/or a combination, and one ormore components.

Exemplary kits can include the microorganisms provided herein, and canoptionally include one or more components such as instructions for use,a device for detecting a microorganism in a subject, a device foradministering a compound to a subject, and a device for administering acompound to a subject.

In one example, a kit can contain instructions. Instructions typicallyinclude a tangible expression describing the microorganism and,optionally, other components included in the kit, and methods foradministration, including methods for determining the proper state ofthe subject, the proper dosage amount, and the proper administrationmethod, for administering the microorganism. Instructions can alsoinclude guidance for monitoring the subject over the duration of thetreatment time.

In another example, a kit can contain a device for detecting amicroorganism in a subject. Devices for detecting a microorganism in asubject can include a low light imaging device for detecting light, forexample emitted from luciferase, or fluoresced from green fluorescenceprotein, a magnetic resonance measuring device such as an MRI or NMRdevice, a tomographic scanner, such as a PET, CT, CAT, SPECT or otherrelated scanner, an ultrasound device, or other device that can be usedto detect a protein expressed by the microorganism within the subject.Typically, the device of the kit will be able to detect one or moreproteins expressed by the microorganism of the kit. Any of a variety ofkits containing microorganisms and detection devices can be included inthe kits provided herein, for example, a microorganism expressingluciferase and a low light imager, or a microorganism expressing greenfluorescence protein and a low light imager.

Kits provided herein also can include a device for administering amicroorganism to a subject. Any of a variety of devices known in the artfor administering medications or vaccines can be included in the kitsprovided herein. Exemplary devices include a hypodermic needle, anintravenous needle, a catheter, a needle-less injection device, aninhaler, and a liquid dispenser such as an eyedropper. Typically, thedevice for administering a microorganism of the kit will be compatiblewith the microorganism of the kit; for example, a needle-less injectiondevice such as a high pressure injection device can be included in kitswith microorganisms not damaged by high pressure injection, but istypically not included in kits with microorganisms damaged by highpressure injection.

Kits provided herein also can include a device for administering acompound to a subject. Any of a variety of devices known in the art foradministering medications to a subject can be included in the kitsprovided herein. Exemplary devices include a hypodermic needle, anintravenous needle, a catheter, a needle-less injection an inhaler, anda liquid dispenser. Typically the device for administering the compoundof the kit will be compatible with the desired method of administrationof the compound. For example, a compound to be delivered subcutaneouslycan be included in a kit with a hypodermic needle and syringe.

G. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Generation of Recombinant Viruses

A Wild type vaccinia virus (VV) strain LIVP (the well known viralstrain, originally derived by attenuation of the strain Lister from theATCC under Accession Number VR-1549, from the Institute of ViralPreparations, Moscow, Russia; see, Al'tshtein et al., (1983) Dokl. Akad.Nauk USSR 285:696-699) designed as VGL was used as a parental virus forthe construction of recombinant viruses designated RVGLX herein. Allvaccinia viruses were purified using sucrose gradient (Yoklik). VVs werepropagated and titers were determined by plaque assays using CV-1 cells(ATCC No. CCL-70). Methods for constructing recombinant vaccinia virusesare known to those of skill in the art (see, e.g., Chakrabarti et al.,(1985 Mol. Cell. Biol. 5:3403 and U.S. Pat. No. 4,722,848)). Table 1summarizes the recombinant VV strains described in this Example.

Inactivation of VV by PUV Treatment

LIVP VV (3×10⁸ pfu/ml) was incubated with 1 μg/ml psoralen (Calbiochem,La Jolla, Calif.), suspended in Hank's buffer at room temperature for 10min, and then irradiated for 5 min in Stratalinker 1800 UV crosslinkingunit (Stratagene, La Jolla Calif.) equipped with five 365 nm long waveUV bulb to produce PUV-VV.

RVGL8: LacZ Insertion into F3 of LIVP

Construction of recombinant vaccinia virus RVGL8 containing a lacZ geneinserted the NotI site was prepared as described in Timiryasova et al.(2001), BioTechniques a 534-540. Briefly it was prepared as follows. TheBamHI/SmaI fragment (3293 bp) of pSC65 (see, Chakrabarti et al. (1997),BioTechniques 23, 1094-1097; see, also Current Protocols in MolecularBiology, Green Publishing and Wiley-Interscience Supplement 15:16.17.2(1992); see also SEQ ID NO: 5 herein and SEQ ID NO: 57 in PCTInternational application No. WO 99/32646) containing the lacZ geneunder the control of the vaccinia p7.5 promoter and strong syntheticvaccinia pE/L promoter was isolated by digestion with restrictionenzymes, blunted with Klenow enzyme, and cloned into SmaI site of pNT8plasmid (Timiryasova et al. (2001), BioTechniques 31: 534-540) toproduce pNZ2 a shuttle plasmid.

To construct pNT8, the NotI region of the wild type VV strain LIVP wasamplified using the following primers:

Forward: (SEQ ID NO: 3) 5′-GGGAATTCTTATACATCCTGTTCTATC-3′; Reverse:(SED ID NO: 4) 5′-CCAAGCTTATGAGGAGTATTGCGGGGCTAC-3′with the VV as a template. The resulting 972 bp fragment containedflanking EcoRI and HindIII sites at the 5′ and 3′ ends, respectively.The PCR product was cleaved with EcoRI and HindIII and inserted in pUC28(Benes et al., (1993) Gene 130: 151. Plasmid pUC28 is prepared frompUC18 (available from the ATCC under Accession Number 37253 byintroducing a synthetic oligo adaptor using primers: pUC28 I:5′AATTCAGATCTCCATGGATCGATGAGCT 3′ (SEQ ID NO: 6); pUC28 II:3′GTCTAGAGGTACCTAGCTAC 5′ (SEQ ID NO: 7) into the EcoRI and SstI sitesof pUC18. This introduces BglII, ClaI, and NcoI sites into thepolylinker of pUC18.

Plasmid pNZ2 contains cDNA encoding the E. coli lacZ gene under thecontrol of the vaccinia virus early/late promoter p7.5 and a syntheticearly/late vaccinia pE/L promoter derived from the plasmid pSC65 (see,Chakrabarti et al. (1997), BioTechniques 23, 1094 1097; see, alsoCurrent Protocols in Molecular Biology, Green Publishing andWiley-Interscience Supplement 15:16.17.2 (1992); see also SEQ ID NO: 5herein and SEQ ID NO: 57 in PCT International application No. WO99/32646). Plasmid pNZ2 provides for homologous recombination of lacZinto the NotI site of the VGL virus (ATCC VR-1549), to produce therecombinant vaccinia virus designated RVGL8. The complex of wild typevaccinia virus DNA digested with NotI and not digested plasmid DNA pNZ2was transfected for in vivo recombination into PUV VV infected cells toproduce RVGL8 (see FIG. 1). RVGL8 and the other recombinant vacciniaviruses described herein are listed in Table 1, below.

Mutant Virus Formation/Transfection

CV-1 African green monkey kidney fibroblasts (ATCC No. CCL-70) grown on60 mm dishes (Corning, Corning, N.Y., USA) were infected with PUV-VV(strain LIVP treated with psoralen and UV; see, e.g., Tsung et al.(1996), J. Virol. 70, 165-171; Timiryasova et al. (2001), BioTechniques11, 534-540; Timiryasova et al. (2001), J. Gene 3 Med. 2, 468-477) atmultiplicity of infection (MOI) of 1.

Two hours post-infection, the cells were transfected with a mixture ofNotI-digested viral DNA (4:g) and intact plasmid DNA (4:g).Lipid-mediated transfection of cells was carried out using 5:1 ofGenePORTER reagent (Gene Therapy Systems, San Diego, Calif., USA) per :gof the DNA according to manufacturers' instructions. Cells wereincubated in transfection mixture for 4 h and then supplemented with amedium containing 20% of fetal bovine serum. Cytopathic effects weremonitored daily by light microscopy. Cells were incubated for 5-7 daysuntil formation of the virus plaques and complete cytopathic effect.Then, infected cells were harvested, resuspended in 0.5 ml of medium,and frozen and thawed three times to release the virus. Single virusplaques were selected for the preparation of small and large recombinantvirus stocks and analyzed for the insertion and expression of the genes.

Confirm Mutant

Viral DNA was analyzed by Southern blots. Briefly, to isolate viral DNA,confluent monolayers of CV-1 cells, grown on 10 cm plates, were infectedwith the wild type VV (strain LIVP) or VV of the virus stock obtainedfrom a single recombinant plaque. When the cytopathic effect wascomplete, cells were harvested and the pellet was resuspended in 3 ml of10 mM Tris-HCl, pH 9.0. Viral particles were lysed, treated withproteinase K, and the virus DNA was isolated by phenol/chloroformextraction, followed by ethanol precipitation. The DNA was resuspendedin 100:1 of sterile water. The viral DNA samples were digested by NotIovernight at 37° C., followed by phenol-chloroform treatment,precipitated and 10 Tg of DNA samples were separated through a 0.8%agarose gel. The DNA was transferred to a positively charged nylonmembrane (Roche Diagnostics Corporation, Indianapolis, Ind., USA) andfixed to the membrane using a GS Gene Linker (Bio-Rad Laboratories,Hercules, Calif., USA). The DIG-labeling of DNA was performed using anonradioactive DNA labeling and detection kit (Roche DiagnosticsCorporation) and incubating for 60 min at 37° C. The membrane washybridized with a denatured DIG-labeled 3357 bp NotI-NotI DNA fragmentof the plasmid pNZ2 encoding the lacZ gene. Hybridization conditions andblot development were performed as suggested by the manufacturer.

The predicted size of the band is 3357 bp. The hybridization of NotIdigested viral DNAs with a 3357 bp DNA probe confirmed the integrationof the lacZ gene into NotI site of virus genome.

Construction of RVGL2 and RVGL23 Viruses with a Single TK Gene Mutation

Vaccinia virus LIVP was used for the construction of recombinant virusRVGL2. Vaccinia virus Western Reserve (WR) was used for the constructionof recombinant virus RVGL23. The cDNA of Renilla luciferase and AequoreaGFP fusion (ruc-gfp; 1788 bp; see, Wang et al., (1996) BioluminescenceChemiluminescence 9:419-422; Wang et al., (2002) Mol. Genet. Genomics268:160-168; Wang et al. (1997) pp 419-422 in Bioluminescence andChemiluminescence: molecular reporting with photons, Hastings et al.,eds., Wiley, Chicheser UK; see, also U.S. Pat. No. 5,976,796; see alsoSEQ ID NO: 8 herein, which sets forth a sequence for a ruc-gfpconstruct) was excised from plasmid pcDNA-ruc-gfp (RG), which isdescribed in Wang et al., (1996) Bioluminescence Chemiluminescence9:419-422 and Wang et al., (2002) Mol. Genet. Genomics 268:160-168 andbriefly below, by restriction endonuclease PmeI and inserted into theSmaI site of pSC65 plasmid (see SEQ ID NO: 5; see, also herein and SEQID NO: 57 in PCT International application No. WO 99/32646), resultingin pSC65-RG-1 plasmid DNA.

Briefly to prepare pcDNA-ruc-gfp, the EcoRI-NotI fragment encoding themodified Renilla luciferase-ending DNA (see, Wang et al. (1997) pp419-422 in Bioluminescence and Chemiluminescence: molecular reportingwith photons, Hastings et al., eds., Wiley, Chicheser UK) was clonedinto the pcDNA3.1 vector (Invitrogen, Carlsbad, Calif.), placingexpression of the Renilla luciferase under control of the CMV promoter.The stop codon at the end of the Renilla luciferase ORF was removed, andthe resulting plasmid digested with NotI. The NotI fragment containingthe ORF encoding humanized Aequorea GFP (Zolotukhin et al., (1996) J.Virol. 70:4646-4654) was excised from the pTR-β-actin plasmid andinserted into the NotI site of the plasmid encoding the Renillaluciferase. The resulting plasmid was designated pcDNA-ruc- the ruc-gfp.

New plasmid pSC65-RG-1 containing ruc-gfp fusion under the control ofthe vaccinia PE/L promoter and E. coli β-galactosidase under control ofp7.5 promoter of VV was used for the construction of a single TK geneinterrupted virus RVGL2 of strain LIVP and RVGL23 of strain WR. CV-1cells were infected with wt LIVP or wt WR virus at MOI of 0.1, and twohours later, pSC65-RG-1 plasmid DNA was transfected using FuGene6transfection reagent (Roche). After 24 h of incubation, cells were threetimes frozen and thawed to release the virus. Recombinant viruses werescreened on CV-cells in the presence of substrate5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside(X-gal, Stratagene,Cedar Creek, Tex., USA). After four cycles of virus purification, allvirus plaques were positive for β-galactosidase expression. Theexpression of the ruc-gfp fusion protein was confirmed by luminescenceassay and fluorescence microscopy, respectively. Schematic maps of theviruses are set forth in FIG. 1.

Construction of RVGL5 and RVGL9 Viruses with Single Gene Mutations

Recombinant vaccinia virus RVGL5 contains the lacZ gene under thecontrol of the vaccinia late p11 promoter inserted into the HA gene ofvaccinia genome (Timiryasova et al. (1993) Mol Biol 27:392-402; see,also, Timiryasova et al., (1992) Oncol. Res 11:133-144.). Recombinantvaccinia virus RVGL9 contains a fusion of the Renilla luciferase gene(ruc) and cDNA of green fluorescence protein (GFP) under the control ofa synthetic early/late vaccinia promoter (PE/L) inserted into the F3gene of the VV genome (Timiryasova et al., (2000)) pp. 457-459 inProceedings of the 11th International Symposium on Bioluminescence andChemiluminescence, Case et al., eds). RVGP5 and RVGLP9 were constructedas described for RVGLP2 and RVGLP23.

Construction of RVGL20 Virus with Double TK and F3 Gene Mutations

The cDNA of human transferrin receptor (hTR) (2800 bp) with polyAsequence was isolated from pCDTR1 plasmid (ATCC Accession No. 59324 and59325) by BamHI, treated with Klenow and inserted into SalI site ofpSC65 plasmid (SEQ ID NO: 5 herein and SEQ ID NO: 57 in PCTInternational application No. WO 99/32646), resulting in pSC-TfR andpSC-rTfR. Plasmid pSC-rTfR contains cDNA hTR in an orientation oppositeto the vaccinia PE/L promoter and E. coli β-galactosidase under controlof the early/late vaccinia p7.5 promoter flanked by vaccinia sequencesfor insertion into vaccinia TK gene. pSC-rTfR was used for theconstruction of RVGL20 virus. RVGL9, a recombinant virus with singledeletion carrying ruc-gfp fusion in the F3 gene locus, which contains aunique NotI site in the LIVP strain (see above, see, also, Timiryasovaet al., (2000) pp. 457-459 in Proceedings of the 11^(th) InternationalSymposium on Bioluminescence and Chemiluminescence, Case et al., eds),was used as a parental virus for the creation of RVGL20 virus byhomologous recombination as described above. A schematic of RVGL20 virusis set forth in FIG. 1.

Construction of RVGL21 Virus with Triple TK, F3 and HA Gene Mutations

The cDNA of the β-glucuronidase (gus) of E. coli (1879 bp) was releasedfrom pLacGus plasmid (Invitrogen; see SEQ ID NO: 9 herein) with XbaI(blunt ended with Klenow fragment) and HindIII, and cloned into pSC11plasmid pSC65 (Chakrabarti et al. (1985) Mol. Cell. Biol. 5:3403-3409;SEQ ID NO: 5 herein and SEQ ID NO: 57 in PCT International applicationNo. WO 99/32646) digested with XhoI (treated with Klenow) and HindIIIunder the control of a vaccinia p11 late promoter, resulting in aplasmid pSC-GUS. The SmaI-HindIII fragment from pSC-GUS plasmid wasinserted into pVY6 plasmid, a vector for inserting antigen genes intothe hemagglutinin gene of vaccinia (see, e.g., Flexner et al., (1988)Nature 355:259-262; Flexner et al., (1988) Virology 166: 339-349; seealso U.S. Pat. No. 5,718,902) digested with SmaI and BamHI, resulting inpVY-GUS plasmid. The resulting plasmid, designated pVY-GUS plasmid,contains the cDNA encoding gus under the control of the vaccinia latepromoter p11 flanked by vaccinia sequences for insertion into thehemagglutinin (HA) gene. Recombinant virus RVGL20 with double deletionswas used as the parental virus for the construction of RVGL21 virus.CV-1 cells were infected with RVGL20 virus at MOI of 0.1. Two hoursafter infection, cells were transected with pVY-GUS plasmid DNA usingFuGene6 transfection reagent (Roche). Recombinant virus plagues wereselected in CV-1 cells by color screening upon addition ofβ-glucuronidase substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronicacid(X-GlcA) (Research Products Int. Co., Mt. Prospect, Ill., USA) into agarmedium. After eight cycles of purification in agar medium in thepresence of X-GlcA pure recombinant virus RVGL21 was selected. RVGL21virus has interruptions of TK, F3 and HA genes and is presentedschematically in FIG. 1.

In Vitro Virus Growth

CV-1, C6 (ATCC No. CCL-107), B16-F10 (ATCC No. CRL-6475), and GI-101A(Rumbaugh-Goodwin Institute for Cancer Research Inc. Plantation, Fla.;U.S. Pat. No. 5,693,533) cells were seeded in 24-well plates at thedensity of 1×10⁵, 2×10⁵, 4×10⁵, and 2×10⁵ cells/well, respectively. Thenext day, the cells were simultaneously infected with 0.001 or 0.01PFU/cell of a wild type LIVP and its mutants. The virus suspension wasadded to cell monolayer (0.15 ml/well) and incubated at 37° C. for 1 hwith brief agitation every 10 min. Then, the virus was removed,appropriate complete growth medium was added (1 ml/well), and the cellswere then incubated at 37° C. for 24, 48, 72 and 96 h after virusinfection. To establish resting cell culture, a confluent monolayer ofCV-1 cells was incubated for 6 days in DMEM with 5 FBS at 37° C. Theseresting cells were infected and harvested at the same time points afterinfection as described above. Virus from the infected cells was releasedby one cycle of freezing and thawing. Viral titers were determined induplicates by plaque assay on CV-1 cells and expressed as PFU/ml.

TABLE 1 List of recombinant vaccinia viruses (VV) Prior InsertionDesignation Designation Description Locus/loci Reference VGL wt VVstrain LIVP No Publicly available VV Insertions RVGL1 recVV2 (p7.5) Luc-HindIII-N- Timiryasova T M, (p11) LacZ of Interrupted Kopylova-SviridovaT N, LIVP VV Fodor I. Mol. Biol. (Russian) 27: 392-401 (1993);Timiryasova T M, Li J, Chen B. Chong D. Langridge W H R, Gridley D S,Fodor I. Oncol. Res. 11: 133-144 (1999) RVGL5 recVV8 (p11) LacZ of HA-Timiryasova T M, LIVP VV Interrupted Kopylova-Sviridova T N, Fodor I.Mol. Biol. (Russian) 27: 392-401 (1993) RVGL7 rVV-EGFP (PE/L) EGFP- TK-Umphress S, Timiryasova or (p7.5) LacZ of Interrupted T., Arakawa T,Hilliker S, rVV-GFP LIVP VV Fodor I, Langridge W. Transgenics 4: 19-33(2003) RVGL8 rVV-Not-LacZ (p7.5) LacZ of NotI (F3)- Timiryasova T M, orLIVP VV Interrupted Chen B, Fodor N, rVV-Not-LZ Fodor I. BioTechniques31: 534-540 (2001) RVGL9 rVV-RG (PE/L) NotI (F3)- Timiryasova T M, YuYa, or Ruc-GFP of Interrupted Shabahang S, rVV-ruc-gfp LIVP VV Fodor I,Szalay A A. Proceedings of the 11^(th) International Symposium onBioluminescence & Chemiluminescence pp. 457-460 (2000) RVGL12 Same asRVGL7, except that HSV TK is inserted in place of gfp RVGL19 (PE/L) TK-and Herein Trf-(p7.5) NotI (F3)- LacZ in Tk Interrupted locus (PE/L)Ruc-GFP in F3 locus of LIVP VV RVGL20 (PE/L) Tk- and Herein rTrf-(p7.5)NotI (F3)- LacZ in TK Interrupted locus (PE/L) Ruc-GFP in F3 locus ofLIVP V RVGL21 (PE/L) Tk-, HA- Herein rTrf-(p7.5) interrupted LacZ in TKand NotI locus, (p11) (F3)- LacZ in HA Interrupted locus, (PE/L) Ruc-GFPin F3 locus of LIVP VV RVGL23 (PE/L) Tk- Herein rTrf-(p7.5) InterruptedLacZ in TK locus of WR VV

Example 2 In Vitro Analysis of Virus Levels

LacZ

Analysis of lacZ expression induced by recombinant vaccinia virus wasperformed as described previously (Timiryasova et al. (2001),BioTechniques 31, 534-540). Briefly, CV-1 cells grown 6-well plates(Corning, Corning, N.Y., USA) were infected with ten-fold dilutions ofthe virus stock. The virus was allowed to absorb for 1 h at 37° C. withoccasional rocking. Then, the virus inoculum was replaced with acomplete medium containing 1% of agar, and the incubation was carriedout for 48 h. To visualize the virus plaques, 300 Tg of X-Gal (MolecularProbes, Eugene, Oreg., USA) per ml and 0.1% of neutral red (Sigma, St.Louis, Mo., USA) were added to the second agar overlay, and plaques werecounted and isolated after 12 h incubation at 37° C. Levels of vacciniavirus in cells in vitro could also be determined by measuring the plaqueforming units (PFU) in the cells.

In Vitro Infectivity of VV's Measured by Plaque Forming Units

The ability of wt LIVP virus and its mutants to infect and replicate wasanalyzed in dividing and resting CV-1 cells as well as in three tumorcell lines (C6, GI-101A, B16-F10). The results demonstrate that vacciniamutants can efficiently infect and replicate in dividing CV-1 cells atan MOI of 0.001. A significant yield of vaccinia virus was obtained fromdividing CV-1 cells. The yield of wt VV and its mutants in dividing CV-1cells was about 10 times higher than in resting CV-1 cells. There was nosignificant difference in viral recovery between vaccinia mutants and wtvirus in vitro studies. The interruption of TK, F3 and HA genes made nodifference to VV mutants replication in the dividing CV-1 cells. Threetumor cells were tested. The relative sensitivities to cytopathiceffects at MOI of 0.001 were follows: CV-1 (dividing, highest), CV-1(resting), C6, GI-101A, B16-F10 (lowest). Mouse B16-F10 melanoma cellswere not sensitive to virus infection at MOI of 0.001. Very low viraltiter was recovered from melanoma cells infected at MOI of 0.01. Alsoobserved was that wt WR strain was able to infect melanoma cells invitro more efficiently compared to LIVP strain and virus recovery washigher compared to LIVP strain.

Example 3 Animal Models and Assays Animal Models

Athymic nude mice (nu/nu) and C57BL/6 mice (Harlan Animal Res., Inc.,Wilmington, Mass.) at 6-8 weeks of age were used for animal studies.Mice in groups of five or four were infected i.v. with 10⁷ PFU of VV ina volume of 0.1 ml i.v. Mice were imaged by low-light imager andfluorescence imager for ruc and for gfp expression, respectively. Thestudy was approved prior to initiation by the Animal Research Committeeof LAB Research International Inc. (San Diego, Calif., USA). All animalcare was performed under the direction of a licensed veterinarian of LABResearch International Inc. (San Diego, Calif., USA).

Glioma Model

To establish subcutaneous glioma tumor, rat glioma C6 cells (ATCC No.CCL-107) were collected by trypsinization, and 5×10⁵cells/0.1 ml/mousewere injected subcutaneously (s.c.) into right hind leg of 6-8 week oldmale athymic mice. On day 7 after C6 cell implantation when median tumorsize was about 150 mm³, viruses at the dose of 10⁷ PFU/0.1 ml/mouse wereinjected intravenously (i.v.). Mice were sacrificed 14 days after virusinjection. In the kinetic studies using of RVGL9 virus, mice weresacrificed at 20 min, 1 h, 4 h, 18 h, 36 h, 3 d, 5 d, 7 d and 14 daysafter virus injection.

Breast Tumor Model

To develop sub cutaneous (s.c). breast tumor, human breast cancer GI-101A cells (Rumbaugh-Goodwin Institute for Cancer Research Inc. Plantation,Fla.; U.S. Pat. No. 5,693,533) at the dose of 5×10⁶ cells/0.1 ml/mousewere injected s.c. into the right hind leg of 6-8 week old femaleathymic mice. On day 30 after GI-101A cell implantation, when mediantumor size was about 500 mm³, viruses at the dose of 10⁷ PFU/mouse wereinjected i.v. Mice were sacrificed on day 14 after virus injection. Micefor survival experiments and breast tumor therapy studies were kept forlong time periods (more than 100 days after virus injection). Mice thatdeveloped tumor with the size about 4000 mm³, and/or lost 50% of bodyweight were sacrificed.

Melanomal Model

For a melanoma model, mouse melanoma B16-F10 cells (ATCC No. CRL-6475)at the dose of 2×10⁵cells/0.04 ml/mouse were injected into the foot padof 6-8 week old male C57BL/6 mice. When the tumor was established(median size of tumor about 100 mm³), on day 18 after cell implantation,viruses at the dose of 10⁷/mouse were injected i.v. Mice were sacrificed10 days after virus injection.

Vaccinia Virus in Animal Models

Vaccinia Virus Recovery from Tumor and Organs of Nude Mice

From sacrificed animals blood was collected, and organs (lung, liver,spleen, kidneys, testes, ovaries, bladder, brain, heart) and tumors wereharvested and homogenized in PBS containing a mixture of proteaseinhibitors. Scissors and forceps were changed after each organdissection or incision to avoid cross-contamination of the tissues.Samples were frozen and thawed, centrifuged at 1,000g for 5 min. Viraltiter was determined in the supernatant diluted in serum-free medium onCV-1 cells by plaque assay and staining them with 1% (wt/vol) crystalviolet solution after 48 h incubation. Each sample was assayed induplicate and viral titer was expressed as mean PFU/g of tissue.

Assay Measurements

Survival studies were performed on 6-week old nude mice bearing s.c.human breast tumor. Mice were injected i.v. with 10⁷ of vaccinia virusesand followed for survival. Individual body weight was measured twice aweek. Gain/loss of body weight after virus infection was calculated asthe percentage: body weight (g)−tumor weight (g) on day of virusinjection/body weight (g)−tumor weight (g) on day of monitoring×100%.Spleens were excised from euthanized animals and weighed. The RSW wascalculated as follows: RSW=weight of spleen (g)×10⁴/animal body weight(g)−tumor weight (g). Mice were euthanized when the mean tumor volumereached 3000 mm³ or developed the signs of disease. Rapid CO₂ euthanasiawas humanely performed in compliance with the NIH Guide for the Care andUse of Laboratory Animals.

Reporter Genes Assays

LacZ

E. coli β-galactosidase activity in tissue samples and in the serum ofthe mice was determined using chemiluminescent Galacto-Light Plus™ Assaysystem (Applied Biosystems, Bedford, Mass., USA) according to theinstructions of the kit manufacturer. Briefly, 1-20 μl of the sample wastransferred into the tube with 200 μl of 1:100 diluted Reaction BufferDiluent and incubated at RT for 30 min. A 300 μl aliquot of accelerator(−II) was added into the tube with the sample, mixed quickly and thesignal was read using luminometer. β-galactosidase activity wasexpressed as relative light units (RLU) per g of tissue. Purified E.coli β-galactosidase (Sigma) was used as a positive control and togenerate a standard curve.

Luciferase

Renilla luciferase activity was measured in the supernatant of thetissue samples after they had been homogenized using a Turner TD 20eluminometer (Turner Designs, Sunnyvale, Calif., USA) as describedpreviously (Yu and Szalay, 2002) with some modifications. In brief, 20μl of the samples was added into 500 μl of luciferase assay buffer (0.5M NaCl, 1 mM EDTA, 0.1 M potassium phosphate pH 7.4) containing asubstrate coelenterazine. Luciferase activity was measured during 10-sinterval and expressed as RLU per g of tissue.

Assay Results

Presence of RVGL9 Over Time

A vaccinia virus RVGL9 with a single F3 gene mutation and carryingruc-gfp was used to assess the pattern of vector tissue distributionfollowing i.v. administration into immunocompromised athymic micebearing s.c. glioma tumors. The tissue distribution data using thisrecombinant virus showed virus distribution and tumor targeting by thisVV strain. Kinetics studies were performed by noninvasive imaging ofvirus replication in the mice based on ruc and gfp expression. Four tofive animals per group bearing s.c. rat glioma C6 tumor were injectedwith 10⁷ of RVGL9 virus via the tail vein. The animals were sacrificedat 20 min, 1, 4, 18 and 36 hours, 3, 5, and 14 days after virusinjection. No viable viral particles were recovered from brain, bladderor testes at any time point after i.v. injection of virus. Some viralparticles were recovered from spleen, heart and lung at early timepoints after virus injection. After 18 h post-infection, the titer ofRVGL9 virus in these organs decreased. No virus was recovered in theheart tissue after 18 h; around 156.5 and 44 PFU/g tissue was recoveredfrom spleen and lung, respectively, on day 14 as compared to 3221.0 and3521.9 PFU/g tissue at 20 min after virus injection, respectively. Thepattern of virus recovery from liver and kidneys was different from thepattern in the spleen, heart, or lung. No virus in the kidneys and 174.9PFU/g tissue of virus was recovered from liver at an early time aftervirus injection. On day 5 after virus injection, the titer of virus inthese organs increased and went down on day 14 post virus injection. Intumor tissue virus was detected starting 18 h after virus administration(1.6×10³ PFU/g tissue), and dramatically increased over the time ofobservation (1.8×10⁸ PFU/g tissue on day 7). Virus in the tumor tissuewas detectable for more then 60 days after a single i.v. virusinjection. The results demonstrate tumor-specific replication of thesevaccinia mutants. A correlation was observed between the virus recoveryand the transgene expression in tumors and in organs. Based on the dataof RVGL9 virus kinetics, day 10 or day 14 was used for tissuedistribution studies of different vaccinia mutants in melanoma andglioma and breast tumor models, respectively.

Presence of Various VV in Mice Bearing a Glioma Tumor

To examine tissue distribution of vaccinia virus in immunodeficient micebearing an s.c. glioma tumor, viruses were injected i.v. at a dose of1×10⁷ PFU/0.1 ml/mouse on day 7 after C6 rat glioma cell implantation.Fourteen days after virus injection, mice were sacrificed and virustiter was determined in different tissues. Mice injected with wt WRvirus were sick and dying due to viral pathogenicity. Hence, WR-injectedmice were sacrificed on day 7 after virus injection. Wild type LIVPvirus was recovered from all analyzed tissues as well as from brain. Theamount of recovered virus particles from the mice injected with wt LIVPwas much lower than wt WR strain of VV. The results are presented inTable 1A.

TABLE 1A Viral recovery from nude mice tissues in glioma model.^(a)RVGL21 LIVP RVGL2 RVGL5 RVGL9 RVGL20 TK−, F3−, WR^(b) RVGL23 Wt TK− HA−F3− TK−, F3− HA− Wt TK−, WR Brain 1.2 × 10³ 1.4 × 10³ 0 0 0 0 1.4 × 10⁷1.9 × 10⁶ Kidneys 6.1 × 10² 6.7 × 10² 1.6 × 10² 34.6 33.3 36.6 5.4 × 10⁶7.9 × 10² Lung 2.9 × 10³ 0 1.6 × 10² 1.4 × 10⁴ 6.7 × 10³ 2.4 × 10³ 1.9 ×10⁶ 2.1 × 10³ Spleen 1.9 × 10² 0 1.8 × 10² 1.0 × 10³ 1.0 × 10² 1.7 × 10²1.6 × 10⁶ 1.8 × 10³ Testes 5.8 × 10⁴ 64.3 6.4 × 10² 7.5 × 10² 0 0 9.8 ×10⁴ 1.7 × 10³ Bladder 6.4 × 10³ 0 0 2.9 × 10³ 0 0 2.8 × 10⁵ 1.2 × 10³Liver 3.4 × 10⁴ 63.6 4.2 × 10² 33.6 96.6 30.8 7.1 × 10³ 5.6 × 10³ Heart6.0 × 10³ 0 0 0 0 0 1.4 × 10⁵ 0 Serum^(c) 0 0 0 0 0 0 6.0 × 10² 0 Tumor5.4 × 10⁷ 1.5 × 10⁷ 3.8 × 10⁷ 2.9 × 10⁷ 3.9 × 10⁷ 1.9 × 10⁷ 1.9 × 10⁸3.7 × 10⁷The results demonstrate that 10000-fold more virus was recovered in thebrain of mice injected with WR strain versus wt LIVP strain. Wild typeWR strain virus was recovered from the serum (600 PFU/20 μl) of mice onday 7 after virus injection. No virus was recovered in the serum of themice injected with LIVP mutants on day 14. The level of wt LIVP in serumwas not tested on day 7. About 1.9×10⁶ PFU/g tissue of TK-mutant of WRstrain (RVGL23) was found in the brain tissue compared to 1.4×10³ PFU/gtissue for mice injected with the TK-mutant of L1VP strain (RVGL2).

All other mutants of VV strain LIVP were found mostly in tumor only andno virus was recovered from brain tissue of mice injected with a doubleor triple mutant (Table 1A). Three times as many virus particles wererecovered from the tumors of mice injected with WR compared to wt LIVP.The mean of viral recovery in tumor tissue of the mutants of LIVP strainwas similar to the wt LIVP and equivalent to TK-mutant of WR strain.

Presence of Various VV in Mice Bearing a Breast Tumor

Data for tissue distribution in immunocompromised mice bearing s.c.GI-101A human breast are presented in Table 1B:

TABLE 1B Viral recovery from nude mice tissues in breast cancer model.RVGL21 LIVP RVGL2 RVGL5 RVGL9 RVGL20 TK−, F3−, WR^(b) RVGL23 Wt TK− HA−F3− TK−, F3− HA− Wt TK−, WR Brain 0 0 0 0 0 0 7.2 × 10⁶ 1.6 × 10⁴Kidneys 3.6 × 10³  38.3 27  3.3 × 10²  25.8 0 3.2 × 10⁷ 2.8 × 10⁵ Lung8.6 × 10³ 5.5 × 10²  29.1 1.6 × 10³ 1.6 × 10³ 1.0 × 10³ 2.1 × 10⁶ 3.7 ×10³ Spleen 5.5 × 10³  99.5 0 1.8 × 10² 0 0 1.6 × 10⁶ 1.8 × 10³ Ovaries1.6 × 10³ 0 0 0 0 0 8.0 × 10⁷ 2.7 × 10⁷ Bladder 3.9 × 10³ 0 0 0 0 0 2.8× 10⁴ 1.2 × 10³ Liver 1.2 × 10⁴ 0 1.7 × 10² 5.2 × 10² 1.7 × 10² 1.0 ×10² 4.0 × 10⁵ 4.8 × 10⁵ Heart 1.4 × 10² 0 0  58.2 4.6 × 10² 0 6.3 × 10⁴2.2 × 10³ Serum^(c) 0 0 0 0 0 0 2.4 × 10³ 0 Tumor 8.6 × 10⁸ 1.0 × 10⁹2.5 × 10⁸ 1.1 × 10⁹ 5.6 × 10⁸ 1.0 × 10⁹ 2.9 × 10⁹ 6.6 × 10⁸About 10-fold more viral particles were recovered from breast tumortissue compared to glioma tumor tissue. No virus particles wererecovered from the brain tissue of mice injected with either wt LIVP orits mutants. 7.2×10⁶ and 1.6×10⁴ PFU/g was recovered from brain tissueof mice injected with wt WR and TK-virus of WR strain VV, respectively(Table 1B). During the dissection of organs from euthanized mice, it wasfound that the ovaries from the mice being injected with wt WR and TK-of WR virus were drastically enlarged as compared to all other groups ofmice. The analysis of viral recovery from ovaries demonstrated hightiter of wt WR and TK-WR strain in ovaries, for example, 8.0×10⁷ and2.7×10⁷ PFU/g, respectively. About 1.6×10³ PFU/g was recovered from theovaries of the mice injected with wt LIVP virus, however no virusparticles at all were recovered from either ovaries or from brain ofmice injected with the mutants derived from LIVP strain (Table 1B).

Presence of Various VV in Mice Bearing a Melanoma Tumor

The tissue distribution of VV in the immunocompetent mice bearingmelanoma tumors on foot pads also were studied. BL/6 mice on day 17after B16F10 melanoma cell implantation were i.v. injected with theviruses at the dose of 10⁷ PFU/mouse via the tail vein. All groups ofmice were sacrificed on day 10 after virus injection due to huge tumorsize in the PBS-injected control group. The results are set forth inTable 1C:

TABLE 1C Viral recovery from C57BL/6 mice tissues in melanoma model.RVGL21 LIVP RVGL2 RVGL5 RVGL9 RVGL20 TK−, F3−, WR^(b) RVGL23 Wt TK− HA−F3− TK−, F3− HA− Wt TK−, WR Tumor 5.4 × 10⁶ 3.9 × 10⁶ 3.7 × 10⁵ 9.5 ×10⁵ 2.5 × 10⁵ 2.4 × 10⁵ 9.9 × 10⁶ 2.2 × 10⁶ Tissues^(e) 0 0 0 0 0 0 0 0^(a)Mean of viral recovery PFU/g of tissue for 3-5 mice/group. ^(b)Micewere sacrificed on day 7 after virus injection. ^(c)PFU/20 μl of serum^(d)Mice were sacrificed on day 9 after virus injection. ^(e)No viruswas recovered in all tested tissue.No virus was recovered from kidneys, lung, spleen, brain, testes,bladder, liver, heart, and serum of the immunocompetent mice injectedwith the viruses. Virus was only recovered from the tumor tissue. About10-fold virus particles were recovered from the tumors of mice injectedwith wt LIVP, TK-LIVP, wt WR, and TK-WR compared to other groups.

Example 4 Reduction of Human Breast Tumor Implanted in Nude Mice byRecombinant Vaccinia Viruses RVGL7, RVGL9 and RVGL21

RVGL7 and RVGL9

FIG. 1 shows a schematic representation of the recombinant vacciniaviruses used for these experiments. RVGL7 was prepared as described forthe preparation of RVGL9. RVGL7 contains nucleic acid encoding EGFP andlacZ, and includes pE/L and p7.5 regulator regions inserted into the TKgene.

Luminescence and Fluorescence Images of Tumors in a Nude Mouse

Human breast GI-101A cancer cells (5×10⁶ cells/mouse) weresubcutaneously implanted into the right thigh of the mice. Thirty daysafter cell implantation RVGL9, the NotI (F3)-interrupted virusexpressing a fusion of Renilla luciferase and green fluorescence protein(RVGL9=rVV-RG=rVVruc-gfp) was injected intravenously via tail vein at adose of 1×10⁷ PFU/mouse. A fluorescence image of GFP and low-light imageof luciferase expression were taken nine days after virus injection,i.e. 39 days post cell implantation showing dissemination of the virus.

Reduction of Human Breast Tumor Implanted into Nude Mice by VacciniaViruses RVGL7 or RVGL9

Human breast GI-101A cancer cells (5×10⁶ cells/mouse) weresubcutaneously implanted into the right thigh of the mice. Mice wereinjected i.v. with RVGL7=rVV-GFT=TK- or RVGL9-rVV-ruc-gfp=NotI(3)-interrupted viruses (1×10⁷ PFU/mouse in 0.1 ml) and PBS control onday 30 after cell implantation. Images were taken on day 65 afterGI-101A cell implantation and 35 days after virus or PBS injection. Theresults demonstrate drastic reduction of tumor volume in the miceinjected with TK- or NotI (F3)-interrupted vaccinia viruses comparedwith the tumor in the mice injected with PBS.

GFP in Human Breast Tumor after Viral Administration

Human breast GI-101A cancer cells (5×10⁶ cells/mouse) weresubcutaneously implanted into the right thigh of the mice. Mice wereinjected i.v. with RVGL7=rVV-GFP=TK- or RVGL9=rVV-RG-rVV-nic-gfp-NotI(F3)-interrupted viruses (1×10⁷ PFU/mouse in 0.1 ml) on day 30 aftercell implantation. The data demonstrate GFP expression in tumor area inthe mice injected with TK⁻ or NotI (F3)-interrupted vaccinia viruses. NoGFP signals were observed in other parts of the mice bodies. The resultsalso showed that expression of GFP can be visualized as early as 48 hafter virus injection through the tail vein. On day 16 after virusinjection very strong signals of GFP which correspond to a tumor volumeof about 1300-1620 mm³ for TK- or NotI (F3)-interrupted virus,respectively were observed. Reduced GFP signals were observed on day 25(1218-1277 mm³ for TK- or NotI (F3)-interrupted virus, respectively) and32 (514-887 mm³ for TK- or NotI (F3)-interrupted virus, respectively)due to reduction of tumor volume.

Time Course of Breast Tumor Volume Over Time

GI-101A breast cancer cells were implanted subcutaneously into the rightthigh of 4-5-week old female athymic (nu/nu) mice in the dose of 5×10⁶cells/mouse. Thirty days after tumor implantation, when the tumorreached about 500 mm³ in volume, a single dose (1×10⁷ PFU/mouse in 0.1ml) of RVGL7=rVV-GFP=TK- or RVGL9=rVV-RG=rVV-ruc-gfp=NotI(F3)-interrupted vaccinia viruses or PBS control was injectedintravenously (via tail vein). Tumor dimensions were measured withvernier caliper twice a week and volumes were calculated as (L×H×W)/2,where L, H and W represent the length, width, and height of the tumor,respectively and expressed in mm³. The data demonstrate significant(60-80% on day 65) tumor reduction in the mice injected with TK-, NotI(F3)-interrupted vaccinia viruses. In contrast, tumors grew very rapidlyin the mice injected with PBS.

Monitoring of Tumor Regression by Light Extinction.

Subcutaneous GI-101A breast tumor reduction occurred in 100% ofimmunocompromised mice treated with a single i.v. injection of wt LIVP,single F3-, single TK-, and double F3-, TK-, mutants of LIVP strain.Some degree of toxicity was seen in the mice treated with the aboveviruses. RVGL21 virus with the triple deletions TK, F3 and HA geneswhich showed no toxicity in nude mice; hence this virus was used forlong-term studies. The difference in antitumor activity and survivalbetween high and low doses of treatment using the triple mutant RVGL21virus was not significant. GFP expression in tumor area in the miceinjected with RVGL21 was monitored. No GFP signals were observed inother parts of the mice bodies. Expression of GFP can be visualized asearly as 48 h after virus injection through the tail vein. On day 16after virus injection we observed very strong signals of GFP, whichcorresponded to tumor volume of about 1300-1620 mm³ and reduced GFPsignals on days 25 (1218-1277 mm³) and 32 (514-887 mm³) due to reductionof tumor volume. Tumor volume reduction also was apparent by visualinspection of the mice.

Example 5 Reduction of Vaccinia Virus Toxicity and Virulence

Reduction of Vaccinia Virus Pathogenicity by Monitoring Mouse BodyWeight and Survival

The percentage of body weight change in athymic and immunocompetent micebearing different s.c. tumors after i.v. administration of the viruseswas examined. Injection of wt LIVP and wt WR and some mutants aethe doseof 10⁷ pfu/mouse via the tail vein led to a progressive vaccinia virusinfection within a two week observation period. At one week afterchallenge, the mice showed typical blister formation on the tail andfootpad. Later, weight loss, sometimes accompanied by swelling of themouth region, in several cases led to death of the mice. In the case ofwt WR strain of VV, mice started to die on day 7 after i.v. injection ofvirus. While mice receiving the recombinant LIVP viruses gained weightor remained the same weight over the same time period.

Body Weight in Glioma Model Nude Mice

Rat glioma C6 cells at the dose of 5×10⁵/0.1 ml/mouse were implanteds.c. into the right thigh of nude mice (5-6 old male mice) on day 0.Vaccinia viruses were injected i.v. (via tail vein) at the dose of 1×10⁷PFU/0.1 ml/mouse on day 7. Animals were weighed twice a week. Gain/lossof body weight on day 14 post infection was calculated as thepercentage: body weight−tumor weight on day of virus injection (g)/bodyweight-tumor weight on day 14 (g)×100%. Injection of VGL (wild typevaccinia virus, strain LIVP) and RVGL5 (HindIII-N-interrupted) causestoxicity in nude mice: mice continue to lose the weight. Recombinantvaccinia viruses RVGL5 (HA-interrupted), RVGL7 (TK-interrupted), RVGL8(NotI(F3)-interrupted), RVGL19 (double, TK- and NotI (F3)-interrupted)were less toxic in nude mice: after losing some body weight, 10 dayspost-infection, mice started to gain the body weight.

Nude mice with glioma that were injected with wild type WR strain of VVlost 31.9% of body weight on day 7 after virus injection. Mice injectedwith TK-virus of WR strain lost 22.4% of body weight on day 14 aftervirus injection compared to 1.5% in the group of mice injected withTK-virus of LIVP strain of VV. All mice injected with wild type LIVPstrain survived for at least 14 days (the duration of the experiment).Mice without tumor injected with VGL (wt VV, strain LIVP) lost 11.23% ofbody weight. Mice bearing tumor injected with VGL (wt VV) or with RVGL I(HindIII-N-interrupted) lost 15.79% and 10.18% of body weight,respectively. Mice in the wt LIVP group lost 15.8% of body weight versus9.4% in the PBS injected group. Tumor-bearing mice injected with RVGL2(TK-), RVGL5 (HA-), RVGL7 (TK-), RVGL8 (F3-), RVGL9 (F3-), RVGL20 (TK-,F3-), RVGL21 (TK-, F3-, HA-) on day 14 after virus injection lost only1.5%, 0.4%, 2.1%, 5.0%, 7.3%, 2.4%, and 3.2% of body weight,respectively. Tumor-bearing mice injected with virus carrying doublegene interruption, RVGL19 (TK- and F3-) demonstrated 0.73% gain of bodyweight compared to the body weight on day 0. Based on the results ofbody weight, a single interruption of HA, TK, F3 (NotI site) and doubleinterruption of TK, F3 (NotI site) genes in vaccinia virus genomereduces virulence and toxicity of the vaccinia virus strain LIVP.

Injection of wt VV strain WR, however, was extremely toxic to nude mice,which died on day 7 after virus injection. Wild type and mutant VVs ofstrain LIVP were less toxic in nude mice. Although nude mice injectedwith various LIVP strains lost some body weight, after day 10-postinfection mice started to gain the body weight.

Body Weight in Breast Tumor Model Athymic Mice

The body weight change of athymic mice with s.c. GI-101A human breasttumor after i.v. injection of vaccinia viruses was monitored. Miceinjected with wt WR strain lost 25.6% of body weight and died due tovirus toxicity. Although mice injected with wt LIVP virus survived forlonger time, mice lost 26.4% of body weight. Mice injected with TK-WRstrain lost 17.8% of body weight, while mice injected with TK-LIVP virusgained 1.9% of body weight. All mice injected with other mutants of LIVPstrain were stable; no virus related toxicity was observed in thesemice.

Body Weight in Melanoma Model Immunocompetent Mice

The toxicity of the vaccinia viruses in immunocompetent C57BL/6 micebearing mouse B16-F10 melanoma on their foot pad was studied. Althoughmice in all groups survived during the experiment, wt WR strain was moretoxic in immunocompetent mice compared to wt LIVP and recombinantstrains. Mice injected with wt WR strain lost about 11.4% of body weighton day 10 after i.v. injection of virus, while mice injected with wtLIVP strain and its double (RVGL20) and triple (RVGL21) mutants lostonly 2.2%, 1.3%, and 0.6% of body weight, respectively, versus to 7.1%of body weight lost in PBS injected mice. Mice administered i.v. withRVGL2 (TK-), RVGL5 (HA-), RVGL9 (F3-), and RVGL23 (TK-WR strain)continued to gain weight over this same period.

Long-Term Survival after Viral Infection for Breast Tumor-Bearing Mice

To examine the effect of different mutations on long-term survival, micebearing s.c. GI-101A human breast tumor received doses of 10⁷ virusi.v., and were observed for survival after viral infection. The resultsshowed that there are differences in survival depending upon the virusinjected. Injection of the nude mice bearing s.c. breast tumor with wtWR strain (i.v., 1×10⁷/mouse) resulted in 100% mortality: four mice offive died on day 9 and one mouse died on day 11 after virus injection.Mice injected with strain LIVP survived for 35 days. Mice injected witha single mutated virus RVGL9 (F3-) developed the toxicity and 25% ofmice died on day 34 after virus injection, however the deletion of F3gene in LIVP strain prolonged the survival of mice up to 57 days. Miceinjected with double mutant virus RVGL20 (F3-, TK-) began to die on day34 after virus injection, but survived longer than F3-injected mice. TheRVGL20 virus injected mice reached 50% survival point on day 65 andshowed significantly longer survival time up to 116 days. The singlemutant TK-virus of LIVP virus was less pathogenic than the single mutantF3-or double mutant F3-, TK-viruses; all mice were alive on day 80 afterinjection with TK-virus and 14.3% of the mice survived 130 days. Allmice injected with the triple mutant TK-, F3-, and HA-virus (RVGL21)survived 130 days (duration of the experiment) and continued to livewithout any signs of virus toxicity compared to other groups of mice.

Splenomegaly in Various Mice

Immunocompetent C57BL/6 Mice

Several groups of the animals demonstrated enlargement of the spleen;therefore the relative spleen weight (RSW) was calculated. The resultsare shown in Table 2 as follows:

TABLE 2 Relative spleen weight (RSW) in mice with or without tumors.Breast cancer Glioma model model Melanoma model Groups nu/nu mice nu/numice C57BL/6 mice No tumor, PBS 43.6 ± 4.1^(a )  50.5 ±11.2^(d)  30.1 ±2.8^(g) No tumor, LIVP 67.2 ± 11.9  48.0 ± 13.1 68.1 ± 9.4 Tumor, PBS92.4 ± 7.4^(b )  84.1 ± 14.6^(e)  106.0 ± 46.1^(h) LIVP  98.2 ± 28.2^(c) 108.4 ± 39.4^(f)  148.4 ± 44.8^(i) RVGL2 96.0 ± 34.9 112.7 ± 15.6 51.9± 6.6 RVGL5 143.8 ± 20.5  169.6 ± 31.7 61.6 ± 2.9 RVGL9 73.9 ± 10.5151.8 ± 27.9  63.3 ± 34.9 RVGL20 84.9 ± 6.6  159.9 ± 22.7 106.7 ± 36.0RVGL21 114.4 ± 12.5  117.7 ± 15.3  63.0 ± 24.6 WR 37.3 ± 3.5   57.9 ±10.9 70.5 ± 1.8 RVGL23 46.9 ± 15.7  73.1 ± 19.3  97.0 ± 43.9 Mean ± SDfor n = 4-8 mice/group. RSW = weight of spleen (g) × 10⁴/(animal bodyweight (g) − tumor weight (g)). ^(a)p ≦ 02.02 vs. all groups, except notumor LIVP, WR, RVGL23 ^(b)p ≦ 0.039 vs. no tumor PBS, no tumor LIVP,RVGL5, WR, RVGL23 ^(c)p ≦ 0.046 vs. all groups, except PBS, RVGL2,RVGL20, RVGL21 ^(d)p ≦ 0.006 vs. all groups except no tumor LIVP, PBS,WR, RVGL23 ^(e)p ≦ 0.048 vs. all groups, except no tumor PBS, LIVP,RVGL2, WR, RVGL23 ^(f)p ≦ 0.045 vs. all groups, except PBS, RVGL2,RVGL21 ^(g)p ≦ 0.035 vs. PBS, LIVP, RVGL20, WR, RVGL23 ^(h)p ≦ 0.049 vs.all other groups, except no tumor LIVP, RVGL20, WR, RVGL23 ^(i)p ≦ 0.049vs. all other groups.As shown in the Table 2 above, some degree of splenomegaly was observedin mice. For immunocompetent C57BU6 mice, a statistically significantdifference (p<0.035) was found in tumorous mice injected with PBS, LIVP,RVGL20, WR and RVG123 compared to non-tumorous mice. In mice injectedwith wt VV strain LIVP spleen was enlarged greatly (p<0.049) versus allother groups. In contrast, the smallest spleens were found in the micewithout tumor.

Nude Mice with a Glioma Tumor

In nude mice with or without s.c. glioma tumor, mice injected with wt WRor TK- of WR virus had the lowest RSW 37.3 or 46.9, respectively, whichwas similar to the RSW from the mice without tumor and injected with PBS(43.6). The largest RSW 143.8 and 114.4 was observed in RVGL5 (HA-) andRVGL21 (TK-, F3-, HA-) groups, respectively. No statisticallysignificant difference was found among the groups of mice injected withwt LIVP, RVGL2, RVGL9, RVGL20 versus the PBS injected group.

Nude Mice with Breast Tumor

The results of RSW in the immunocompromised mice bearing s.c humanbreast tumor indicate that all mice injected with wt LIVP and itsmutants have an enlarged spleen compared to the mice injected with wt WRor TK-WR viruses (p<0.045). The largest spleen was found in the miceinjected with single HA-, single F3-, double F3-, TK-mutants of LIVPstrain.

Other Results Using RVGL21 for Injection

Two mice, #437 and #458, survived more then 190 days after RVGL21injection (10⁷ and 4×10⁵, respectively, i.v.) without any signs ofdiseases or virus related toxicities.

On day 30 after GI-101A cell implantation (tumor volume=594.9 mm³), 10⁷of RVGL21 was injected i.v. into mouse #437. On day 101 after virusinjection (s.c. tumor size=220.4 mm³), metastasis (hard tissue) in chestarea under the skin was observed. The size of the tumor was 1223.6 mm³,which disappeared by day 148. The s.c. tumor did not disappear, itstarted to grow back, but the mouse remained metastasis-free.

Mouse #458 had a first s.c. tumor (GI-101A) on the right hind quarter.When the first tumor started to shrink (day 29 after RVGL21 virusinjection, tumor size=1924.3 mm³), a second syngeneic tumor wasimplanted s.c. on the left hind quarter. The second tumor grew slowly,reached the size of 1205.7 mm³ and started to shrink. The mouse was freeof the first tumor on day 127 post virus injection; the size of thesecond tumor was 439.6 mm³. The tumor continued to shrink and the cellsdied. The body gradually absorbed remaining tumor tissues that werecontributed by the host (such as the tumor vascular skeleton that wascoming from the host). Since these remains are not considered foreign,the immune system doesn't destroy them. The tumor cells, on the otherhand, were long gone and cleared by the immune system and the virus.Reduction of the second syngeneic tumor demonstrates that this mousedeveloped antibodies against the tumor cells. The antibodies resulted inthe reduction of the second syngeneic tumor.

Example 6 Use of a Microorganism or Cell to Induce Autoimmunization ofan Organism Against a Tumor

This example shows that the method provided herein and in priorityapplication EP 03 018 478.2 relating to “The production of apolypeptide, RNA or other compound in a tumor tissue” also can be usedfor the production of antibodies against the tumor tissue. Theseantibodies provide for autoimmunization of the organism bearing thetumor. Furthermore, these antibodies can be isolated and used for thetreatment of tumors in other organisms.

Methods and uses of microorganisms, including cells, which can containDNA encoding a desired polypeptide or RNA, to induce autoimmunization ofan organism against a tumor are provided. Also provided are methods forthe production of antibodies against a tumor by: (a) injecting amicroorganism, such as a virus or cell, optionally containing a DNAsequence encoding a desired polypeptide or RNA, into an organism bearinga tumor and (b) isolating antibodies against the tumor.

This Example further demonstrates that administration of microorganisms,such as the triple mutant vaccinia virus strain provided herein, whichaccumulate in tumors, causing them to release tumor antigens for asufficient time to permit production of antibodies by the host. This isexemplified by showing a reduction and elimination of xenogeneic GI-101Asolid breast carcinoma tumors and their metastases in nu−/nu− mice (Tcell deficient mice).

Step#1: Female nu−/nu− mice of 5 weeks age were chosen, and the GI-101Acells grown in RPMI1640 medium, supplemented with estrogen andprogesterone. The confluence was reached, cells were harvested, washedwith phosphate buffered saline. Cells (5×10⁶ cells per mouse) were theninjected subcutaneously into mice. The tumor growth was carefullymonitored every two days.Step#2: At two stages of tumor growth (at tumor size of 400-600 mm³, andat tumor size of ˜1700 mm³), purified vaccinia viral particles (RVGL12)were delivered to each tumorous mice by intravenous injection throughtail vein. The colony purified virus was amplified in CV-1 cell line andthe intracellular viral particles were purified by centrifugation insucrose gradient. Two concentrations of virus (10⁶ pfu/100 μl and 10⁷pfu/100 μl resuspended in PBS solution) were injected. The viralreplication was monitored externally by visualization of virus-mediatedgreen fluorescence protein expression. The tumor development wasmonitored by tumor volume determination with a digital caliper.

Vaccinia viruses RVGL12+GCV(gancyclovir), and RVGL12 (RVGL12 is the sameas RVGL7, except that the nucleic acid encoding gfp is replaced byherpes simplex virus thymidine kinase (HSV TK; see, SEQ ID NOS: 35 and36) were injected 67 days after GI-101A cellular implantation. A secondadministration referred to as RVGL12a, was injected 30 days aftercellular implantation.

Step#3: After viral administration, it was determined that first thetumors continued to grow to a size of ˜900 mm³ (from 400-600 mm³ at thetime of viral injection), and to a size of ˜2400 mm3 (from 1700 mm³).Then the growth rate leveled off for approximately 6-8 days.Step#4: Approximately 14 days after viral injection, the tumor volumestarted to decline rapidly. Forty days after viral application, all thetreated animals showed more than 60% tumor regression. Sixty-five daysafter viral treatment and many of the animals had complete regression oftumors.Step#5: Some of the animals were completely tumor-free for several weeksand their body weight returned to normal. RVGL-12+GCV treatment resultedin 86.3% reduction of tumor size (Day 52 after viral injection) fromtheir peak volumes on Day 13, RVGL-12 treatment resulted in 84.5%reduction of tumor size (Day 52) from their peak volumes (Day 13).RVGL-12a treatment resulted in 98.3% reduction of tumor size (Day 89)from their peak volumes (Day 12). After PBS+GCV control treatment, theaverage volume of tumors were increased by 91.8% in 38 daysStep#6: The level of immune activation was determined. Sera wereobtained from the animals with regressing tumors and the immune titerdetermined against a foreign protein (e.g. green fluorescent protein),vaccinia viral proteins, and GI-101A cancer cell proteins weredetermined. The following antisera obtained from the following sourceswere used to analyze the following listed samples.

Samples:

1). Mouse cell lysate (control);2). Purified and denatured vaccinia viral particles;3). GI-101A tumor cell lysate;4). Purified green fluorescent protein;5). Purified luciferase protein;6). Purified beta-galactosidase protein.

Antisera:

a). Antiserum from nontumorous mouse;b). Antiserum from GI-101A tumorous mouse;c). Antiserum from GI-101A tumorous mouse 14 days after vaccinia i.v.injection;d). Antiserum from GI-101A tumorous mouse 65 days after vaccinia i.v.injection;e). Antiserum from tumor-free mouse (after elimination of GI-101A tumor)80 days after vaccinia i.v. injection.

The results showed that there was enormous tumor-specific vaccinia virusreplication in the tumors, which led to tumor protein antigen and viralprotein production in the tumors. In addition, the vaccinia virus didlyse the infected tumor cells thereby releasing tumor-cell-specificantigens. The continuous leakage of these antigens into the body led toa very high level of antibody titer (in approximately 7-14 days) againstforeign cell proteins (tumor proteins), viral proteins, and the virusencoded engineered proteins in the mouse body. The newly synthesizedantitumor antibodies and the enhanced macrophages, neutrophils countswere continuously delivered via the vasculature into the tumor andthereby providing for the recruitment of an activated immune system inthe inside of the tumor. The active immune system then eliminated thetumor including the viral particles. This interconnected release offoreign antigens boosted antibody production and continuous return ofthe antibodies against the tumor-contained proteins function as anautoimmunization vaccination system, initiated by vaccinia viralreplication, followed by cell lyses, protein leakage and enhancedantibody production.

Example 7 Production of β-Galactosidase and Anti β-Galactosidase ViaVaccinia Virus Delivered lacZ in Tumor Bearing Mice

Thirty five athymic nu/nu mice (5 weeks old, 25 g, male) were used todemonstrate the biodistribution and tumor targeting of vaccinia virus(strain LIVP) with different deletions in the genome. Mice were dividedinto 7 groups with 5 in each group as presented in Table 1

No. Virus Insertion Group mice Tumor implanted Injected locus 1 5 NoneVGL wtLIVP 2 5 C6, s.c. 5 × 10⁵ cells VGL wtLIVP 3 5 C6, s.c. 5 × 10⁵cells RVGL1 N-luc, lacZ 4 5 C6, s.c. 5 × 10⁵ cells RVGL5 HA- lacZ 5 5C6, s.c. 5 × 10⁵ cells RVGL7 TK-egfp, lacZ 6 5 C6, s.c. 5 × 10⁵ cellsRVGL8 NotI-lacZ 7 5 C6, s.c. 5 × 10⁵ cells RVGL19 TK-rTrf, lacZ, NotI-RGC6 gliomas were subcutaneously developed in Groups 2 to 7. Five daysafter tumor cell implantation (5×10⁵ cells/mouse), each animal wastreated with 0.1 ml of virus at a multiplicity of infection (MOI) of1×10⁷ via tail vein injection. Two weeks after virus injection, all micewere sacrificed and blood samples were collected. Various organs andtumors also were taken from animals for virus titer and β-galactosidaseanalysis.

The β-galactosidase analysis was performed using the Galacto-Light Plussystem (Applied Biosystems), a chemiluminescent reporter gene assaysystem for the detection of β-galactosidase, according to themanufacturer's instructions.

β-Galactosidase Expression Measurements

In non-tumorous mice as well as in tumorous mice injected with wild typevaccinia virus (without reporter genes and without β-galactosidase gene)no β-galactosidase expression was detected in organs, blood and tumorsamples. By contrast, in the tumors of mice infected withβ-galactosidase expressing virus, high levels of β-galactosidase wasexpressed. β-galactosidase also was detected in blood samples as shownin Table 3, but no virus was recovered from blood samples.

TABLE 3 Production of β galactosidase by vaccinia virus in tumor andblood from tumor bearing mice (day 14 after virus injection) β-gal intumor Est. total β- Est. total β- Virus Tg/mg of total β-gal in serumgal/tumor gal/5 ml Group Injected protein Tg/ml of total protein (Tg)blood (Tg) 3 RVGL1 1.59 ± 0.41 1.38 × 10⁻² ± 1.09 × 10⁻² 489.84 4.00 4RVGL5 1.51 ± 0.37 1.16 × 10⁻² ± 1.08 × 10⁻² 330.21 3.62 5 RVGL7 1.35 ±0.59 0.95 × 10⁻² ± 1.47 × 10⁻² 616.60 1.83 6 RVGL8 1.81 ± 0.42 0.86 ×10⁻² ± 0.33 × 10⁻² 962.36 2.38 7 RVGL19 1.30 ± 0.44 0.26 × 10⁻² ± 0.16 ×10⁻² 463.75 0.60

Anti-β-Galactosidase Antibody Production

To determine whether the amount of β-galactosidase presented in mouseblood was sufficient to elicit antibody production, sera taken from twomice (mouse #116 from Group 5, and #119 from Group 6) were collected andtested for primary antibodies against β-galactosidase in Westernanalysis. β-galactosidase from E. coli (Roche, 567 779) was used as theantigen standard, and the mouse monoclonal anti β-galactosidase from E.coli (Sigma, G6282) was used as the antibody positive control. Asadditional sources of β-galactosidase, total protein was obtained fromCV-1 cells 24 hours after infection with RVGL7 at MOI of 1 pfu/cell, andthe tumor protein sample from mouse designated #143 (treated with RVGL7)was obtained.

The protein samples were prepared in triplicate, each set including aβ-galactosidase antigen control, a cell lysate from RVGL7 infected CV-1cells, and tumor lysate from mouse #143. All protein samples wereseparated by electrophoresis using a 10% polyacrylamide gel, andtransferred to NitroBind nitrocellulose membrane (MSI) using a BioRadsemidry blotting system. Immunoblotting was performed with either 1:3000mouse monoclonal anti β-galactosidase, or 1:3000 mouse serum taken fromeither mouse #116 or #119, and 1:3000 Goat AntiMouse IgG-HRP (BioRad).An Amplified Opti-4CN Detection Kit (BioRad) was used for detection.

The results showed that sera taken from mouse #116 and #119 exhibitedsimilar levels of antibody when compared to a commercial mouseanti-β-galactosidase standard, and demonstrated that the tumor bearingmice #116 and #119 produced antibodies against β-galactosidase.

Example 8 Mammalian Cells for Tumor Therapy

As shown herein, certain bacteria, viruses, and mammalian cells (BVMC),when administered systemically, again enter and selectively replicate intumors Hence, systemically injected mammalian cells and certainbacterial (anaerobic bacteria, such as Salmonella, Clostridium sp.,Vibrio, E. coli) cells gain entry into solid tumors and replicate intumor-bearing organisms. Genetically-labeled cells can be used for tumordetection and therapy. In addition to gene expression in tumors throughBVMC targeting, tumor-specific gene expression can be achieved bylinking transgenes to tissue/tumor-specific promoters. To obtain tumorspecific gene expression, a variety of systemic targeting schemes can beemployed. These strategies include the use of tissue/tumor-specificpromoters that allow the activation of gene expression only in specificorgans, such as prostate-specific promoter-directed viral geneexpression; the use of extracellular matrix (i.e. collagen)-targetedviral vectors; and the use of antibody-directed viral vectors.Conditionally-replicating viruses have also been explored astumor-specific delivery vehicles for marker genes or therapeutic genes,such as oncolytic adenovirus vector particles, replication-selectiveHSV, vaccinia viruses and other such viruses.

When light-emitting protein encoded BVMC are injected systemically intorodents, tumor-specific marker gene expression is achieved and isdetected in real time based on light emission. Consequently, thelocations of primary tumors and previously unknown metastases in animalsare revealed in vivo. Hence diagnosis can be coupled to therapy and tomonitoring of therapy. The impaired lymphatic system in tumors may beresponsible for the lack of clearance of bacteria from tumors by thehost immunosurveillance after escaping the vascular system.

Example 9 Tumor Development is Inhibited Following S. pyogenesAdministration

This example and following examples demonstrate the use of bacterialcells to colonize tumors, use of reporter in the cells to quantitatecolonization; use of the colonized attenuated bacterial cells for tumorinhibition. Co-administration or sequential administration of bacteriaand viruses. Administration of virus before bacteria increase tumorcolonization by the bacteria. Administer bacteria that expresses anenzyme that will activate a prodrug, thereby targeting colonized cells.

Bacterial Strains

Streptococcus pyogenes M-type 1 T-type 1 (ATCC catalog no. 700294) wastransformed with pDC123-luxF plasmid) that contains the bacterialluciferase expression cassette (Lamberton G R, Pereau M J, IIles K,Kelly I L, Chrisler J, Childers B J, Oberg K C, Szalay A A. 2002.Construction and characterization of a bioluminescent Streptococcuspyogenes. Proceedings of the 12th International Symposium onBioluminescence and Chemiluminescence, Case J F, Herring P J, Robison BH, Haddock S H D, Kricka U. Stanley PE (eds). Chichester: Wiley, pp85-88. Luciferase can be detected in the presence of exogenous decanal.

Transformed S. pyogenes were grown overnight in BH1 media in thepresence of 20 μg/ml of chloramphenicol at 37° C. After overnightgrowth, the bacteria were counted at OD₆₀₀ and bacteria were resuspendedin BH1 media at the indicated density for injection.

Tumor Development and Bacterial Injection

Twenty 5-week old mice were injected subcutaneously in the right lateralthigh. Each mouse was injected with 5×10⁵ C6 glioma cells transformedwith pLEIN-derived retrovirus (Clontech; see also WO 03/14380). Thesubcutaneous tumors were developed for 7 days after implantation beforebacterial injection.

For bacterial injection, the tumor-bearing mice were anesthetized withisofluorane. The suspensions were injected intravenously with a 1-ccinsulin syringe equipped with a 29½-gauge needle through a surgicallyexposed femoral vein. After the injections, the incisions were sutured.

Tumor growth was monitored twice a week following bacterial injectionusing a digital caliper. In addition, fluorescence imaging andphotographic images of the animals were taken at the end time points.The presence of luminescent bacteria was analyzed by intravenouslyinjecting the animals with 30 μl of decanal. Analysis of whole animalsfor bacterial luciferase activity, followed methods similar to Yu et al.(2004) Nature Biotechnology 22(3): 313-20. Briefly, anesthetized animalswere placed inside the dark box for photon counting (ARGUS 100 low lightImager, Hamamatsu). Photon collection was for 1 minute from ventral anddorsal sides of the animal and the images were recorded with Image ProPlus 3.1 software (Media Cybernetics) and/or Lighttools® macroimagingsystem. A light image also was recorded. The luminescent images weresuperimposed on the light image to localize the luminescent activity onthe animal. Total intensity of photon emission in localized regions,e.g. in the tumor region, also was recorded. S. pyogenes was isolatedfrom removed tumors and ground tissue was plated on LB-chloramphenicol(20 μg/ml) plates. Luminescent bacteria were counted in the presence ofdecanal vapor.

Results

Four groups of mice were tested. Each group contained five mice.

Group S. Pyogenes 1 None 2 1 × 10⁶ 3 1 × 10⁷ 4 5 × 10⁷Tumor volume was measured after 7 days of tumor development and theinjection of S. pyogenes, through 21 days post-tumor development.

The control group of mice with no S. pyogenes had continuous andaccelerating tumor growth over the 2-week period. The mice injected withS. pyogenes had slower tumor growth. Groups 3 and 4 had the slowesttumor growth rates. Both groups maintained a slower linear ratethroughout the monitoring period, whereas the control group, notinjected with bacteria, exhibited tumor growth that accelerated at latertime periods.

At all time points following bacterial injection, tumor volumes weresmaller in Groups 3 and 4 mice than in the control mice (Group 1). Atday 21, the average tumor volume of the control group was approximately2.5-3 fold greater than the average tumor volumes in Groups 3 and 4.Group 2, injected with the lowest titer of bacteria, also had a reducedtumor volume from the control group at the later time points, althoughthe tumor volume was larger than Groups 3 and 4.

Bacterial colonization and tumor inhibition also is assayed in afibrosarcoma model. HT1080 fibrosarcoma cells transformed with the pLEINretrovirus are injected subcutaneously into the right lateral thigh offive week old nude male mice 5×10⁵ cells/mouse). S. pyogenes transformedwith pDC123-luxF is injected into the femoral vein of the animals after8 or 14 days of tumor growth (5 animals on each day). A group of 5animals are not injected as serve as a control group. Tumor growth andluciferase activity is monitored at subsequent time points. S. pyogenesis isolated from tumors and cultured on BH1+chloramphenicol (20 μg/ml)plates. Luminescent bacterial colonies are counted in the presence ofdecanal vapor.

Example 10 Vibrio Cholera Localization to Tumors Plasmids and BacterialStrains

Attenuated Vibrio Cholerae, strain Bengal 2 serotype 0139, M010 DattRS1,was transformed with pLITE201 which contains the IuxCDABE cassette(Voisey et al. (1998) Biotechniques 24:56-58). The transformed strain isa light emitting strain due to the expression of the luciferase genes.

Tumor Development and Bacterial Injection

Groups of nude mice (n>20) were implanted with C6 glioma tumors (500mm³) as described in the Examples herein. 1×10⁸ transformed bacteria (V.Cholerae) were suspended in 100 μl of phosphate buffered saline (PBS).The bacterial suspension was injected into the right hind leg of eachmouse. The animals were then monitored after injection under a low lightimager as described in Example 3.

In a separate experiment, for comparison, groups of nude mice (n>20)were implanted with C6 glioma tumors (500 mm³) as described in theExamples herein. These mice were injected with 1×10⁸ pfu/mouse ofrVV-RUC-GFP (RVGL9) virus (see Example 1).

Results

Titer and Luciferase Activity

Mice from each of the two injected groups were sacrificed at time pointsafter injection. Tumors were excised and homogenized. Bacterial andviral titers and luciferase activities were measured as described in theExamples herein.

Both bacterial and viral titer increased following injection. Theincrease in bacterial growth over time was proportional to luciferaselevels in the tumors. A log-log plot of bacterial titer versusluciferase activity in tumors in the mice injected with V. cholerademonstrated a linear relationship between bacterial titer andluciferase activity. The groups of mice injected with rVV-RUC-GFP virus,also demonstrated a linear relationship between virus titer andluciferase activity.

Time after V. Cholera/pLITE injection 4 hrs 8 hrs 16 hrs 32 hrsBacterial Titer 3.79 × 10⁴ ± 2.93 3.14 × 10⁶ ± 2.45 1.08 × 10⁸ ± 1.35.97 × 10⁸ ± 4.26 (cfu/tumor) Time after rVV-ruc-gfp virus injection 36hrs Day 3 Day 5 Day 7 ViralTiter 3.26 × 10⁶ ± 3.86 7.22 × 10⁷ ± 3.671.17 × 10⁸ ± 0.76 3.77 × 10⁸ ± 1.95 (pfu/tumor)The experiments demonstrated a linear relationship between titer andluciferase activity. Thus, luciferase activity of the injected bacteriaand/or virus can be used a correlative measurement of titer.

Localization

Localization of V. cholera was performed as detailed in the Examplesherein for virus. Briefly, organs and blood samples were isolated fromanimals euthanized with CO₂ gas. The organs were ground and plated onagar plates with chloramphenicol drug selection for analysis ofbacterial titer.

Bacterial titer was assayed in tumor, liver, testes, spleen, kidney,lung, heart, bladder and brain of the injected mice. Samples were takenfrom mice sacrificed at zero, and subsequent times up to 150 hoursfollowing V. cholera injection.

At the time point immediately following injection (t=0), V. cholera waspresent in all samples, with the highest levels in the liver and spleen.By 50 hours post-injection, titer of V. cholera in all tissues hadreduced with the exception of tumor tissue. In contrast, V. choleratiter had increased about 4 orders of magnitude as compared to timezero. This level increased slightly and then stayed constant throughoutthe remainder of the experiment. By 150 hours post-infection, titer inall samples except tumor had decreased. For example, the titer in liverhad decreased by approximately 5 orders of magnitude from the time zeropoint. At the 150 hour point, the V. cholera titer in the tumor tissuewas about 6 orders of magnitude greater than any other tissue sample.

Example 11 Co-Administration and Sequential Administration of Bacteriaand Virus

V. Cholera/pLITE (see Example 10) and vaccinia virus RVGL2 (seeExample 1) were administered together or sequentially. Groups of nudemice with C6 glioma tumors were injected with bacteria and/or virus asshown in the Table below. Three male mice were injected per group.Bacteria and/or virus were injected on day 11 and day 16 following tumorimplantation. Tumor growth, luciferase and GFP activity were monitoredas described in the Examples herein.

Group Day 11 injection Day 16 injection 1 1 × 10⁷ VV-TK⁻-gfp-lacZ 1 ×10⁷ V. Cholera/pLITE 2 None 1 × 10⁷ V. Cholera/pLITE 3 1 × 10⁷ V.Cholera/pLITE 1 × 10⁷ VV-TK⁻-gfp-lacZ 4 None 1 × 10⁷ VV-TK⁻-gfp-lacZ 5None 1 × 10⁷ VV-TK⁻-gfp-lacZ and 1 × 10⁷ V. Cholera/pLITE

Results

On day 21 (21 days post tumor implantation) animals were sacrificed.Tumors were excised from each animal and ground. Viral titer was assayedon Groups 3, 4 and 5. Bacterial titer was assed on Groups 1, 2 and 5.Titers (colony forming units and plaque forming units) were performed aspreviously described in the Examples.

A comparison of the bacterial titer in tumors Groups 1, 2 and 5demonstrated that bacterial titer was highest in Group 1 that had beeninjected first with vaccinia virus at day 11, and followed by V. cholerainjection on day 16. Co-injection of bacteria and virus at day 16 (Group5) gave an intermediate bacterial titer. Group 2, injected only with V.cholera at day 16, had a lower bacterial titer in the tumor tissue thaneither of groups 1 or 5. Thus, tumors were more susceptible to bacterialcolonization when first colonized by VV-TK⁻-gfp-lacZ virus.

A comparison of the viral titer in Groups 3, 4 and 5 demonstrated thatGroup 4, with only virus injection at day 16, had the highest viraltiter followed by Groups 5 and 3. The viral titer of Group 5 wasslightly higher than Group 3, but not apparently significantlydifferent. One mouse in Group 4 had a viral titer that was an extremeoutlier in comparison to the viral titer of the other 2 mice in Group 4.When the numbers were reassessed without this mouse, the general trendremained the same. The average viral titer in Group 4 was much closer tothe viral titers of Groups 3 and 5. The data from the three groups inthis analysis was not significantly different. Thus, pre-administrationof bacteria followed by administration of virus did not significantlychange the viral colonization of the tumor as compared with viraladministration alone.

Example 12 Tumor Inhibition by Administering PNP-Expressing Bacteria andProdrug Plasmids

pSOD-DeoD contains the bacterial purine nucleoside phosphorylase gene(PNP) (Sorcher et al. (1994) GeneTher. 1(4):223-238), under the controlof the constitutive SOD (superoxide dismutase) promoter. PlasmidpSOD-DeoD-lux, contains the luxCDABE expression cassette (Voisey et al.(1998) Biotechniques 24:56-58) inserted into pSOD-DeoD.

PNP converts the non-toxic prodrug 6-methylpurine deoxyribose (6-MPDR)to 6-methyl purine which inhibits DNA replication, transcription andtranslation (Sorcher et al. (1994) GeneTher. 1(4):223-238).

Tumor Growth Inhibition

Nude mice were injected with pLEIN retrovirus transformed C6 gliomacells. The pLEIN retrovirus expresses EGFP under the control of theviral promoter LTR (Clontech; see also WO 03/14380). E. coli DH5αexpressing the bacterial purine nucleoside phosphorylase gene wasinjected at day 8 following tumor implantation with or without prodrug(6-methylpurine deoxyribose (6-MPDR)). Tumor volume was monitored atsubsequent time points (as performed in previous examples).

Group Administered 1 E. coli/PNP + prodrug 2 E. coli/PNP 3 E. colicontrol + prodrugGroups 2 and 3 exhibited equal tumor growth over time points from 8 to21 days post tumor implantation. Group 1, which received both the E.coli expressing PNP and the prodrug exhibited ˜20% reduction in tumorsize as compared to the control Groups 2 and 3 at the end time points.

To further test bacterial colonization and prodrug effects on tumorgrowth, a human breast cancer model, GI-101A adenocarcinoma in nudemice, was chosen. GI-101A was derived from GI-101. GI-101 originatedfrom a local first recurrence of an infiltrating duct adenocarcinoma(stage IIIa, T3N2MX) in a 57 year old female patient by researchers atRumbaugh-Goodwin Institute for Cancer Research. In the subcutaneousxenograft nude mice model, the tumor consistently metastasizes to thelungs. The GI-101A is a slower growing tumor model as compared to the C6glioma tumor model.

Fifteen 4 week old female nude mice are each injected subcutaneously inthe right lateral thigh with GI-101A cells. Thirty days after tumordevelopment, bacteria are injected. Escherichia coli DH5α is transformedwith pSOD-DeoD or pSOD-DeoD-lux. The bacteria are grown overnight in LBmedia in the presence of 20 μg/ml of chloramphenicol at 37° C. Afterovernight growth, the bacteria are counted at OD₆₀₀ and bacteriaresuspended in BH1 media at the indicated density. The suspensions areinjected intravenously with a 1-cc insulin syringe equipped with a29½-gauge needle into the animal through a surgically exposed vein or asotherwise indicated. After the injections, the incisions are sutured.

Prodrug is administered to groups of mice every four days followinginjection of bacteria. Tumor growth is monitored twice per week using adigital caliper. Luciferase imaging is performed as described in theExamples herein. At the end point, the animal are sacrificed and organsare assayed as described in Example 9. Histological analyses areperformed to determine the degree of tumor necrosis due to bacterialcolonization and/or drug treatment.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A recombinant vaccinia virus, comprising a modified thymidine kinase(TK) gene and a modified hemagglutinin (HA) gene, and an insertion,deletion or mutation in the gene or locus designated F3, wherein: eachof the TK, HA and F3 gene or locus is inactivated; and the F3 locusoccurs on the HindIII-F fragment of the vaccinia virus between openreading frames F14L and F15L.
 2. The vaccinia virus of claim 1, whereinheterologous nucleic acid is inserted into the TK and HA genes toinactivate them, and heterologous nucleic acid is inserted into the F3locus.
 3. The method of claim 1, wherein the vaccinia virus is a strainselected from among Western Reserve (WR), Copenhagen, Tashkent, TianTan, Lister, Wyeth, IHD-J, and IHD-W, Brighton, Ankara, MVA, Dairen I,L-IPV, LC16M8, LC16MO, LIVP, WR 65-16, Connaught and New York City Boardof Health.
 4. The vaccinia virus of claim 1, wherein the insertion inthe F3 locus is at a site corresponding to the unique NotI or position1475 of the LIVP strain inside of the HindIII-F fragment of the LIVPstrain of vaccinia virus.
 5. The vaccinia virus of claim 1, wherein atleast one of the TK, HA gene and F3 locus comprises an insertion ofheterologous nucleic acid that encodes a protein.
 6. The vaccinia virusof claim 5, wherein the protein is an antibody or antigen-bindingfragment thereof.
 7. The vaccinia virus of claim 5, wherein the proteinis wherein the gene encodes a solute carrier protein (slc), a superoxidedismutase (sod), a colony stimulating factor (csf), monocyte chemotacticprotein-1 (mcp-1), a nonmetastatic protein (nm), a neurogenin protein(ngn), erythropoietin (epo), bmp2, bmp2a, bmp4, tyrp1, tyrp2 or aim 1.8. The recombinant vaccinia virus of claim 7, wherein the heterologousnucleic acid encodes an slc selected from among solute carrier 5 (slc5)and solute carrier 6 (slc6) transporter protein families.
 9. Therecombinant vaccinia virus of claim 8, wherein the transporter proteinis a norepinephrine transporter (net) or is a sodium-iodide symporter(nis).
 10. The vaccinia virus of claim 5, wherein the heterologousnucleic acid is operatively linked to a regulatory sequence.
 11. Thevaccinia virus of claim 10, wherein the regulatory sequence comprises apromoter.
 12. The vaccinia virus of claim 10, wherein the regulatorysequence comprises the vaccinia virus early/late promoter p7.5.
 13. Thevaccinia virus of claim 10, wherein the regulatory sequence comprises anearly/late vaccinia pE/L promoter.
 14. The vaccinia virus of claim 5,wherein heterologous nucleic acid encodes a detectable protein or aprotein capable of inducing a detectable signal.
 15. The vaccinia virusof claim 5, wherein the heterologous protein is an anti-tumor agent oran anti-metastatic agent.
 16. A pharmaceutical composition, comprisingthe virus of claim
 1. 17. The pharmaceutical composition of claim 16that is formulated for systemic administration.
 18. The pharmaceuticalcomposition of claim 16 that is formulated for intravenousadministration.
 19. A method for eliminating immunoprivileged cells inan animal, comprising administering the pharmaceutical composition ofclaim 16 to the animal, whereby the virus accumulates in theimmunoprivileged cells, thereby resulting in elimination of the cells ora reduction in their number.
 20. The method of claim 19, wherein theimmunoprivileged cells comprise tumor cells.
 21. The method of claim 19,wherein the vaccinia virus encodes a detectable protein or protein thatinduces a detectable signal; and the method further comprises detectingthe detectable protein or detectable signal inside the subject tothereby detect tumor tissue and/or metastases and/or to monitor therapy.22. The method of claim 19, wherein the vaccinia virus is a strainselected from among Western Reserve (WR), Copenhagen, Tashkent, TianTan, Lister, Wyeth, IHD-J, and IHD-W, Brighton, Ankara, MVA, Dairen I,L-IPV, LC16M8, LC16MO, LIVP, WR 65-16, Connaught and New York City Boardof Health.
 23. The method of claim 19, wherein the animal is selectedfrom among humans, gorillas, monkeys, rodents, fowl, ruminants, deer,sheep, pigs, horses, cats, dogs, and rabbits.
 24. The method of claim19, wherein the animal is a human.
 25. A method for imaging tumor cellsor tissue in a subject, comprising administering a virus of claim 1,wherein the virus encodes a detectable protein or protein that induces adetectable signal, to detect virus accumulating in the subject andthereby detect tumor cells in which the viruses accumulate.
 26. A methodof treatment of cancer, comprising administering the pharmaceuticalcomposition of claim 16 to a subject for treatment of cancer, whereintreatment comprises an alleviation, reduction or amelioration ofclinical symptoms or diagnostic markers associated with the tumor. 27.The method of claim 26, wherein the cancer comprises a solid tumor. 28.A method of treatment of cancer, comprising administering a combinationof a pharmaceutical composition of claim 16, and an anti-cancertreatment or agent to a subject for treatment of cancer, whereintreatment comprises an alleviation, reduction or amelioration ofclinical symptoms or diagnostic markers associated with the tumor. 29.The method of claim 28, wherein the cancer comprises a solid tumor. 30.The method of claim 26, wherein the virus is a Lister strain virus. 31.A combination, comprising: a virus of claim 1; and a chemotherapeutic oranti-tumor agent.